Center for Drug Evaluation

　　NMPA

　　Apr-2023

I. Preface

　　In 2017, the Former China Food and Drug Administration issued the Technical Guidance for Research and Evaluation of Cell Therapy Products (Trial ), which comprehensively elaborated on the technical requirements for cell therapy products developed in accordance with relevant laws and regulations on drug management. With the advance of stem cell technology, the deepening of cognition and the accumulation of experience, the technical requirements of relevant products have also been gradually revised and improved. This guidance is intended to further standardize the application of stem cell products, guide the CMC research and development of stem cell products, and promote the development of stem cell products industry.

　　The guidance provides suggestions on the technical issues of pharmaceutical research of stem cell products developed as drugs based on the existing understanding. In addition to this guidance, providing scientific and reasonable basis, applicants/holders may also adopt other effective methods and means according to the actual situation of stem cell product research and development, on the premise of meeting the law of drug research and development.

II. Scope

　　The guidance mainly provides technical guiding for the CMC researches at the stage of new drug application, NDA, of human stem cell products subject to develop and registration application in accordance with relevant regulations for drug management.

　　“Human stem cell products” in this guidance refer to stem cell products or stem cell derived products obtained after a series of in vitro operations involving stem cells, generally including expansion, gene modification, induced differentiation, and transfer (differentiation), added into pharmaceutical adjuncts, dispensed into specific containers, and meet specific drug release standards, and they can be directly applied or combined with tissue engineering materials for clinical therapeutic products. These products are originating from human adult stem cells (ASCs or adult cells), human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) humans. Due to the involvement of human cells, the cells for production should meet the relevant provisions of national ethics.

　　Since human-derived stem cell products may involve various technical requirements, relevant guidelines for other drugs, cell therapy and gene therapy may be referred while referring to the Guidelines. The Guidelines do not involve products of reproductive cells, hematopoietic stem cell transplantation and others.

III. General Considerations

　　Stem cells have the potential to self-renew and multilineage differentiate. The cell types of stem cell products are diverse, and the product cells themselves have the ability to live, proliferate and/or differentiate, and interact with cells in vivo. The process of stem cell products production generally includes the acquisition, transportation and acceptance of donor materials, establishment and verification of cell lines/banks, production, inspection, release, storage and transportation. In the stem cell products development, process design and validation, quality study and control, stability study of stem cell products, the stem cell characteristics in the steps above should be fully considered.

　　Stem cell products should follow the general rules of drug research and development. The stage of clinical trials application shall meet the requirements for safety, and the preparation of samples in trials shall meet the requirements of Good Manufacturing Practice - Appendix for Drugs used in Clinical Trials; the relevant studies in process and product quality shall be improved continuously during the clinical trials period; in the application files for NDA, the complete study data for supporting the safety, effectiveness and controllable quality of products should be presented.

A. Biological Activity

　　The biological activity is a key quality attribute of stem cell products, involving the evaluation on aspects of product quality, efficacy, inter-batch quality consistency, comparability study and stability. The study on biological activity should run throughout the whole life cycle of preclinical, early clinical, pivotal clinical and marketing and post-marketing studies. For the study on the biological activity of various products from different sources, it is necessary to make specific analysis according to the characteristics of various stem cell products. For example, the following should be considered: specific markers are continuously discovered and updated; the screening and identification methods suitable for large-scale production in the process are continuously explored and become increasingly efficient and standardized; the differentiation efficiency and homogeneity between product batches need to be improved; the validation strategy of functional cells is not the same, so that the corresponding product release acceptance criteria lack a reliable basis. In the process of setting product biological activity standards, product attributes shall be widely studied, sufficient product characterization data (i.e., molecular, biochemical, immunological, phenotypic, physical and biological characteristics) shall be collected, scientific research progress and clinical findings shall be tracked, and active efforts shall be made to explore which product attributes are most related to efficacy, so as to ensure that the batches of products meeting the determined quality standards or acceptance limits are acceptable to patients and ensure the safety and effectiveness of products.

B. Tartget Differentiation and Unexpected Biological Effects

　　When targeting differentiation strategy is used for the production of stem cell products in vitro, inducing or inhibiting materials and their doses, addition sequence, maintenance time and endpoint residues will significantly affect the differentiation efficiency, and bring quality and safety risks, especially the risks caused by impurities such as unintended cells during the induction of differentiation, the risk of unexpected changes in cells after processing, and the risk of unexpected biological effects caused by the local microenvironmental impact on migration to the body at the same time. Therefore, in the R & D and production process of stem cell products, attention should be paid to the monitoring of the differentiation stage of products, such as genetic markers of germ layer, markers of stemness maintenance in gene-edited hematopoietic stem cells, etc., to determine a reasonable marker detection system, ensure the directional differentiation of final products, and prevent and control the unexpected biological effects caused by non-directional differentiation.

C. Tumorigenicity and Oncogenicity

　　Stem cell products may have high risks of potential tumorigenicity and oncogenicity. The related risk factors include: (1) complex preparation process, long-term cell passage and operation of stem cells; (2) undifferentiated cells, unexpected differentiated cells, malignant transformed cells and mutations that may remain in stem cell products; (3) genetic and epigenetic variation/instability during cell culture; (4) high-risk gene elements that may be introduced by gene modification and oncogene activation or tumor suppressor gene inactivation caused by gene/viral vector insertion. In order to control the above risks, the tumorigenicity and oncogenicity of stem cell products should be assessed and controlled with the combination of the above factors.

D. Residual Pluripotent Stem Cells

　　Cell therapy products derived from ESCs/iPSCs may pose a risk of teratoma, other mispatterned tumors, or precancerous lesions due to a small number of residual pluripotent stem cells or differentiated progeny cells that continue to proliferate. These risks suggest effective control through ESCs/iPSCs cell line selection, efficient targetting differentiation, strict purification, etc., and focus on the development of sensitive detection methods for the detection of contamination of differentiated pluripotent stem cells in the final product (especially when providing local high-density high-dose cells). It is encouraged to evaluate the potential possibility of tumorigenesis through karyotyping, comprehensive analysis at the genome-wide and epigenetic group levels, and risk analysis.

E. Genetic and Epigenetic Studies

　　For stem cell products with complex in vitro operations and pluripotent stem cell derived products, it is recommended to evaluate the genetic and epigenetic stability of in-process samples or final products at appropriate stages in combination with cell characteristics (such as gene mutation analysis, deacetylation detection, etc.). Analytical methods and evaluation criteria applicable to the genetic and epigenetic assessment of stem cell products are evolving to assess the limitations of any such test and to weigh the risk/benefit and the number of patients in any particular case. In some cases, the risks of genomic mutations of stem cell products and genetic disease-related gene mutations, etc., can be analyzed and controlled sequencing methods.

F. Study on Genetically Modified Cells

　　For the development of gene modification process, the gene modification method, element design and operating conditions shall be clarified, and the transduction/transfection mode and efficiency of gene substances shall be clarified after full study. Evaluation should be performed on the integration situations/integration characteristics of the target gene in the genome (insertion site, copy number of the insertion, abnormal growth of the dominant clone, etc) and the function of the target gene expression product. The targeting of gene editing and the expression efficiency of gene substances in the target cell population should be fully studied. In-depth analysis and study should be performed on the chromosomal structural stability, genomic stability, genetic and expression stability of target gene, off-target editing, residual amount of vector for transduction/transfection and virus replication ability reverse mutation of modified cells. Other effects of genetic modification, such as changes to cell viability, stemness, phenotype, function, characteristics, process-related impurities and non-target cell populations, should also be considered.

G. Other Common Issues that Should be Concerned

　　Stem cell products could have the common quality risk factors as other cell therapy products, such as: (1) significant diversities of cells from different donors and different tissue sources of even the same donor, and the differences in donor age, health, cell lineage between tissue sources and target therapeutic tissues, all above may have great impact on product quality. (2) Materials of human-derived/animal originated that may have to be added during cell culture, and adventitious agents that may be introduced during the operation, of all above have the risks of contamination and cross-contamination. For allogeneic products, more attention should be paid to the risk of introduction or transmission of exogenous agents. (3) Autologous products have limited batch size and insufficient test materials. (4) For non-frozen fresh preparations, the shelf life of the product is short. (5) Limited accumulation of product batch data and limited knowledge of critical process parameters (CPPs) and critical quality attributes (CQAs). (6) Multiple rounds of process changes may occur to the products, and the product quality cannot be completely and accurately characterized. For these risk factors, the following considerations should be considered for the control strategy:

　　(1) In terms of production environment, site clearance and isolation operating specifications should be established, and continuous and closed equipment and facilities are encouraged to use to reduce the environmental exposure links in the production process.

　　(2) For raw materials, the qualification and applicability evaluation of donor materials and cell banks, should be consistently conducted. Donor screening should be scientific and reasonable. The alternative cell banks to be re-produced should be concerned. The stability study of cell banks and process intermediates should be carried out. The suppliers of other raw materials should be evaluated and reviewed. Raw materials of human-derived/animal origin that have to be used, should be strictly controlled.

　　(3) In terms of production process and process control, the effective traceability system should be established. Cell identification testing during production should be conducted. The batch number and identification of upstream and downstream materials should be well controlled to prevent confusion and errors. The analytical testing of cell banks and unprocessed harvest fluid should be focus. In the process control key points, relevant test items of samples, such as sterility, endotoxin, mycoplasma, endogenous and exogenous viruses, should meet the requirements. Reasonable in-process control indicators and waste indicators should be defined. The verification or quality monitoring on process intermediates should be well conducted.

　　(4) In terms of quality study and quality control, the concept of quality by design is also applicable to stem cell products. The characterization of products and processes, as well as the development of analytical detection methods should be carried out as early as possible. The product should be comprehensively analyzed for quality attribute and data collection. The critical quality attributes of products should be defined with scientific bases. The comprehensive and effective quality control strategy should be employed in combination with applicable and reliable analytical methods to ensure stable and consistent product quality. Because such products are often difficult to characterize precisely, release testing may be supplemented by enhanced control of raw materials, in-process controls, and testing of process intermediates. In some cases, some novel, validated, rapid and miniaturized release test methods may be used to aid traditional testing methods. The manufacturing process development, transfer and validation should be conducted as requirements.

　　Changes in raw materials and processes often occur during development or even after approval. On the basis of knowledge accumulation on the relationship between processes and quality in the early stage, with understanding the possible impact of changes in raw materials and processes on product quality, systematic comparability study should be carried out for the changes, and quality characteristics study should be carried out continuously in combination with product and process characteristics to fully characterize cell morphology, activity, genetic stability, tumorigenicity/oncogenicity, cell phenotypic characteristics (including expected and unexpected cell populations, etc.) and biological activity, with particular attention paid to the detection of stem cell characteristics affecting product quality (if applicable).

　　(5) In terms of stability, representative batches and samples should be used to carry out the study. Some sensitive indicators, such as cell survival rate and biological activity, should be monitored in the stability study. Accumulated multi-batch data are helpful to understand the trend of product quality and provide the basis for product specifications and shelf life, as well as comparability evaluation.

IV. Materials for Production

　　Production materials include the raw materials, excipients and consumables used in the production process. Their sources and qualities should be clear and reliable. Risks from the introduction or transmission of adventitious agents from materials in production should be minimized. The suppliers of materials and contract manufacturers shall be evaluated and reviewed, and the responsible subjects shall be identified to ensure the quality of materials.

A. Raw Materials

　　Raw materials include starting materials (cells for production, production helper cells, in vitro gene modification systems, etc.) and other raw materials (such as culture media and cytokines). The quality of raw materials is directly related to the quality of final products. The risk assessment and quality control are carried out based on the principles of science and risk, and the compliance with the requirements of “Quality Control of Raw Materials and Excipients for Production of Biological Products" and "Preparation and Quality Control of Animal Cell Substrates for Production and Testing of Biological Products" in the current edition of Chinese Pharmacopoeia.

1. Starting Raw Materials

1.1 Cells for production

　　Cells for production may be directly from a donor (autologous or allogeneic) or from a cell bank. The sources of cells for production should be stable with consistent qualities. The source and related operations of the cells for production should comply with the related national laws and regulations and ethical requirements. The related parties should be informed. The consent should be obtained. The "informed and confidential" management system should be established.

1.1.1 Donor screening

　　Donor screening is an important means of risk control of stem cell product quality. Establishing reasonable donor screening procedures and standards is necessary to prevent risks such as viral contamination, tissue rejection, and related diseases. The relevant characteristics of donors should be collected as far as possible by screening, including but not limited to general information (such as age, gender, blood type, health status, living habits), past medical history and family history (especially infectious diseases and genetic diseases), medication history and radiation exposure. In donor screening, it also needs to distinguish the risk differences between autologous donors and allogeneic donors for different considerations and requirements.

　　For the screening of pathogenic microorganism infection in donors, especially for allogeneic donors, relevant requirements for blood collection and supply, such as screening donors for HBV, HCV, HIV, Treponema pallidum and other infections, can be adopted. In actual products development, appropriate screening items should be added according to the specific circumstances of donor cell type, donor health/disease history or residence in regional epidemic areas, and cell-/tissue-specific pathogens, and included in the list of acceptance criteria. For example, for the live cells and tissues donors with rich white blood cells, it is recommended to screen for HTLV-1 and HTLV-2, CMV, etc. For autologous donors, the screening items and standards may be adjusted appropriately according to the characteristics of the tissues or organs from which the donors are derived, the characteristics of stem cell products and clinical indications, etc., it should be determined whether the production process will increase the risk of pathogen transmission from which the donors become the source of infection, and the chosen preventive measures (in order to prevent the transmission of viruses or other exogenous agents to others than autologous recipients) should be explained. In order to ensure the reliability of pathogen screening results, blood screening kits approved by regulatory authorities should be the prioritized options to detect pathogens for donor screening. In the absence of blood screening reagents, approved in vitro diagnostic reagents can be used. The applicability of detection methods should be confirmed. Attention should be paid to the detection capability of method sensitivity to pathogenic microorganisms. In the absence of approved detection methods or products, self-built methods with comprehensive methodological verification can be used. When universal stem cell products are prepared, the effect of window period on the screening of donor pathogenic microorganism infections must be considered.

　　For the risks of tissue rejection, the relevant requirements for blood transfusion and organ transplantation donors, such as collecting the ABO blood group, Rh blood group, HLA-I and II typing information of donors, can be adopted. It is suggested to consider the tissue matching between donor and recipient according to the product type and clinical application requirements. In case of risk of tissue rejection and high immunogenicity, tissue matching compatibility should be tested in clinic. For non-national standard methods, the test methods and basis should be provided.

　　It is recommended to fully evaluate the potential risk of disease introduction in recipients in combination with advanced analytical detection techniques, such as detection of disease-causing genes in allogeneic donors.

　　In order to develop stem cell products derived from cell banks, screening of donors of this cell bank should be extended, for example, the detection objects of umbilical cord stem cell donors are recommended to be extended to include maternal peripheral blood within appropriate time intervals and umbilical cord blood of the fetus on the day of tissue collection.

1.1.2 Donor tissues and cells

　　The acquisition of donor tissues should follow the standard procedures of acquisition of human tissues and routine protective measures to prevent and reduce the risks of introduction or transmission of endogenous and exogenous factors as far as possible. The procedures such as acquisition, storage, transportation and incoming inspection organized by suppliers shall be fully studied. Effective traceability systems shall be developed. The standard operating procedures and key quality control parameters shall be clarified. The verification, if necessary , shall be completed. When the donors are collected to enter the sites, the detection or confirmation in terms of tissue type, quantity and microorganism shall be carried out according to the process requirements and product characteristics.

　　The isolation process of cells from donor tissues may have an impact on stem cell product quality. It is recommended to use the representative donor-derived tissues in the development of isolation process, and pay attention to key quality characteristics such as cell identification, survival rate and growth activity, exogenous pathogenic microorganisms and stem cell characteristics detection (such as cell surface marker populations, expression products and differentiation potential, etc.) during the treatment. When the primary cells from donor tissue isolation and the directly collected donor cells reach the sites, or the isolated cells after preliminary expansion, the cell type, quantity, phenotype, viability, microorganisms, induced differentiation potential and other aspects should be correspondingly detected according to the process requirements and product characteristics. For example, the cell type can be identified and confirmed by relevant biomarkers (proteins, genes). The proportion of cells positive (or negative) for the marker can be used as the justification for the assessment of the expected population or non-target population index.

1.1.3 Cell system establishing and banking

　　In order to ensure the lot to lot consistency of product quality, it is recommended to establish cell banks for process. It is suggested to follow the guidance of ICH Q5D: Sources and Identification of Cell Substrates Used for Production of Biotechnological Products and Biological Products, Preparation and Verification Procedures for Animal Cell Substrates Used for Production and Verification of Biological Products in Chinese Pharmacopoeia, to carry out cell lines establishing, banking and verifying for suitable cells and cell seeds for production (Cell Seed), with consideration on cell characteristics and process demands. For human embryonic stem cells and induced pluripotent stem cells, especially genetically modified pluripotent stem cells, cell lines should be monoclonal after screened and established, and should be banked.

　　Cell banking should follow the guidance and requirements of current Good Manufacturing Practices. The banking frequency of commercial scale shall be determined by the product characteristics and product batch scale. In cases of banking the cells from multiple donors, such as mesenchymal stem cell products, it is necessary to pay special attention on quality consistency of donor materials to ensure the quality uniformity within and between the cell banks. If banking happens in the pluripotent stem cell stage, such as allogeneic ESCs/iPSCs-derived stem cell products, the cell banks should be comprehensively verified. The contents of ESCs/iPSCs cell bank test generally include cell morphology, identification, activity, markers, cell stemness/pluripotency (such as detection of pluripotency genes OCT4, SOX2, NANOG, etc., detection of differentiation markers in the direction of three germ layers, teratoma test), differentiation potential, microbiological safety, impurity residues, genetic stability (chromosome stability such as karyotype or digital karyotype analysis, whole genome sequencing, whole exome sequencing) and epigenetic studies. In the case of monoclonal banks of genetically modified pluripotent stem cells, it is necessary to evaluate the genomic stability of cell banks, for that, the entire genome sequencing and other methods could be employed to verify the function and safety analysis of modified genes for cell banks. The relevant requirements can follow the section "(VI) Study on Genetically Modified Cells" in the section "III. General Considerations" in this guidance, except for that the study on transduction/transfection mode and efficiency of gene substances is not applicable. In the situations of that library construction and production are not unsuitable, such as autologous gene-modified hematopoietic stem cell products, the solid evidences should be provided to support that the quality control strategy is sufficient to ensure the quality consistency between product batches.

1.1.4 Genetic stability

　　Stem cells may be unstable during passage, producing heterogeneous cells, and high-passage stem cells may have safety risks, so whether bank stem cells or non-bank stem cells, sufficient and standardized studies on genetic stability must be conducted. It is recommended to define the cell passage in terms of population doubling level (PDL) or to clarify the population doubling time (PDT) during cell passage in genetic stability studies.

　　The conditions for genetic stability study shall be representative to actual clinical/commercial production process, with focus on genetic stability, tumorigenicity/oncogenicity, cell stemness, totipotency and target cell differentiation capacity. The limit passage number for in vitro production and the passage number for clinical use shall be established and specified according to the study. The study items of passage stability generally include cell morphology, STR identification, survival rate, number of viable cells, population doubling time, phenotype and other growth characteristics stability, but also genetic stability such as karyotype and genomics sequencing, and also stability of stemness such as polyfunctional gene expression, the formation of teratoma, and directional induction of differentiation.

1.2 Helper Cells for Production

　　If auxiliary cells (such as virus packaging cells and feed layer cells) are used in the production process, the necessity and rationality of their use should be fully explained. Auxiliary cells should comply with the basic principles of clear traceability of sources, controllable safety risks and grading management of established cell banks. It is necessary to clearly trace the source of auxiliary cells, culture and system establishing/banking, analyze, evaluate and detect and control the risks of introducing exogenous factors or immunogenicity. The added amount in process and residual amount of auxiliary cells in the products should be studied and verified. For the processes that may involve cell inactivation treatment, such as irradiation or addition of drugs, it should be proven that it will not affect the safety and function of the product. Ancillary cells can be banked and comprehensively tested according to the requirements for cell bank testing in the Pharmacopoeia. It is recommended to pay special attention to the testing of human-derived/animal-derived viruses. The stability of helper cell bank cells of different generations should be investigated in combination with the characteristics and functions of helper cells.

1.3 In Vitro Gene Modification System

　　For stem cell products directly produced by in vitro gene modification, or stem cell products derived from induced pluripotent stem cells (human induced pluripotent stem cells are obtained through exogenous gene expression, compound induction, epigenetic modification and other routes using human adult cell initiation) produced by reprogramming technology, the upstream production mostly involves in vitro gene modification system. For pharmaceutical professional technical requirements such as design, preparation and quality control of gene modification system (including virus packaging cells), Guidance for Pharmaceutical Research and Evaluation Techniques of In-Vitro Gene Modification Systems (Trial) can be reference.

2. Other Raw Materials

　　The scientific rational and safety of the use of raw materials, as well as the continued accessibility during large-scale production, need to be fully considered. The raw materials used in production shall be clearly indicated with source, composition, usage, dosage and quality control, and shall have the raw material source certificate, inspection report, package insert, TSE/BSE risk analysis and other documents. It is recommended to give priority to the raw materials with high quality grade and low risk. If research-grade reagents (such as culture medium, cytokines, chemical small molecules, etc.) are used in the production, they should be produced in accordance with GMP requirements as far as possible in order to ensure product consistency and purity. If the reagents are not produced in accordance with GMP requirements have sufficient research data should be presented to support the reagents quality high enough for use in humans. Beta-lactam antibiotics such as penicillin, etc., should not be used in cell product process. For raw materials that may affect product safety, if it is assessed to be no impacts on product safety eventually, residues of them are still need to be assessed and controlled at the appropriate stage of the final product or production, and the residue limits should be determined in combination with the clinical dose effect. Some high-risk raw materials may require a separate safety assessment in animals.

B. Excipients

　　For the relevant requirements for excipients, see "(I) Process Development 2.2 Excipients" in the section of "V. Manufacturing Process".

C. Contacting Consumables, Containers and Drug-Device Combination

　　Contact consumables and containers refer to the consumables and containers in direct contact with process intermediates in the production process (disposable tubing, cell factory, biological reaction bag, filter, etc.). Suitability and biosafety assessment shall be carried out in correspondence with product characteristics, and relevant studies shall be carried out. For drug-device combination products, the interaction and risk between components and samples, as well as the effect of components on the function of stem cell products, should be investigated.

V. Manufacturing Process

　　Stem cell products are diverse in types. Different cell types and their preparation process are also different in content and process. Development and process of these products should follow the general law of drug production process, apply the concept of quality by design, pay attention to the study on the relationship between production process and product quality, strengthen the study and validation on impurity removal, and establish a robust and reliable production process.

A. Process Development

　　The production process of stem cell products generally includes upstream drug substance process and downstream drug product process. The process generally includes multiple process steps such as acquisition, recovery, passage or expansion, activation or pretreatment, gene modification, induced differentiation, purification, harvest, filling, freezing and transportation of cells. The boundaries of drug substance and drug product may sometimes be unclear and need to be appropriately divided according to the product situation. By process development and process characterization, process parameters and their range (especially the critical process parameter (CPP) and key process parameters (KPP)) can be setup, as well as production process control, acceptance criteria and waste criteria, the definition of production scale, batch definition and batch size can be clarified. Upstream and downstream process scale should match with each other. The process should be relatively stable and reliable. In principle, different batches of drug substances of human stem cell products should not mix to develop into as drug products. If a scaled-down model is used for process studies, the representativeness of the scaled-down model should be confirmed.

1. Drug Substance Process

　　Drug substance process development may focus on cell culture processes, induced differentiation processes, genetic modifications and other processes, as well as purification processes. During the design of manufacturing process, unexpected or abnormal changes in cells should be avoided and the requirements for removal of related impurities and corresponding functions shall be met.

1.1 Cell Culture Process

　　The quality attributes of stem cell products are generally formed at the cell culture stage. Long-time cell culture could face certain risks. These risks could be: (1)There is a risk of contamination of cultured cells with pathogens in vitro. (2) During cell culture, prolonged passage may lead to accumulated mutation, genomic and epigenetic instability, thereby changing cell differentiation ability or function, especially because mutant cells may have a growth advantage in culture. Therefore, during the process development process, the appropriate culture system (culture medium and addition factor) and culture mode should be strictly screened and controlled, and the culture time and culture generation should be accurately controlled. For example, in the development of non-feed layer cell expansion processes, use suspension culture, micro-vector based three-dimensional culture methods for large-scale expansion, and use stem cells within passage generations supported by passage stability studies to prepare final products.

1.2 Induced Differentiation Process

　　By inducing differentiation, cells should have corresponding cell biological attributes to support the function intended for clinical application. In the process of directional induction of differentiation, it is necessary to combine the development or differentiation process of stem cells with induction and regulation on stem cells together to turn the stem cells into the target functional cells. In order to effectively control the occurrence of non-target differentiation of stem cells, it is necessary to use appropriate inducers or inhibitors to improve the efficiency of directional. The production process should be able to effectively regulate cell fate at different differentiation stages and control that terminal cells no longer to differentiate or trans-differentiate unexpectedly. The composition, purity, stemness, multipotency, differentiation potential, marker expression, and biological functions (differentiation ability and cytokine secretion profile) of the cell population at each stage can be selected at appropriate differentiation points to be monitored.

1.3 Genetic Modifications and Other Processes

　　Some process strategies, such as genetic modification, cell activation, or pretreatment, may help enhance the desired biological activity of stem cell final products. Among them, specific therapeutic functions can be enhanced by initiating or overexpressing specific therapeutically active proteins through genetic modification. Cytokine stimulation, chemical induction, physical intervention (such as hypoxic treatment), etc., can improve immune regulation. When these process strategies are adopted, relevant cell function validation should be performed and the safety risk introduced should be fully assessed.

1.4 Purification Process

　　The purpose of the purification process is to maximize purity, i.e. to increase the ratio of cells with a specific phenotype in total cells and to reduce non-target cells and other impurities in the final cell products. Development studies and process characterization should be performed in the purification process to assess purification efficiency and yield for each purification steps and purification process parameters.

　　Due to the complexity, components of non-target cells may be cells from different or the same lineage, incompletely differentiated cells, or unneeded cells, such as undifferentiated stem cells. Purity analysis of the products should combine the analysis and identification of each component of process intermediates, clarify the properties of each component as completely as possible, and analyze the characteristics of non-target cells.

2. Drug Product Process

2.1 Drug Product Formulation

　　For the selection of dosage form, it is necessary to consider such factors as the clinical application of product, the stability and viability of storage and transportation. The formula from design and screening should be suitable to the dosage form and can effectively maintain the activity and stability of products. The final drug product and use method should be determined after serious studies, like, whether it is of fresh cells or cryopreserved cells, whether it is in the form of free cells or bound to the matrix, whether it is directly administered after cryopreserved cell resuscitation or given to patients after washing and other process treatments. At the same time, it is also necessary to clarify the formulation composition, drug product strength, cell concentration, etc.

2.2 Excipients

　　Through risk assessment and sufficient formulation screening study and verification, the scientific rational and safety of the excipients are proved. The source, quality and dosage of the excipients are specified. The excipients in the formulations should comply with compendial requirements. It is recommended to select excipients as low-risk as possible. Pharmaceutical grade excipients should be the prioritized options. Appropriate nonclinical safety studies should be conducted when using novel excipients as the requirements of relevant guidances.

2.3 Drug Product Manufacturing Process

　　The process of drug product should be determined with the study of product characteristics, formulation and dosage form. In the process development, the concept of quality by design can be applied, with attention on the preparation method, process operation time, filling accuracy and aseptic condition control of the formulation. If the preparations need to be cryopreserved, studies on the cryopreservation and resuscitation process of the preparations should be carried out. It is encouraged to carry out studies on different cooling procedures using advanced equipments with on-line monitoring for cooling. For products stored frozen or otherwise, the effect of short-term or long-term storage on product activity and stability should be determined.

3. Process In-process Controls

　　Stem cell products are complex, and product quality is difficult to be completely and accurately characterized. The concept of quality by design can be applied to strengthen process control. In order to control the consistency of batch quality of products, appropriate process control should be setup for the whole process of stem cell product process, including clear process parameters and in-process standards, to ensure its quality (consistency, purity and efficacy) and safety. These process parameters include the culture conditions (temperature, dissolved oxygen, pH, CO2) of specific steps, amount of process additives used, time control range of each link in production, etc. At the same time, the testing of process intermediates can be conducted at reasonable procession points to detect the process performance indicators related to product quality, such as cell morphology, viability, phenotypic characteristics (including expected and unexpected cell populations), bioburden, recovery rate, etc., and acceptance criteria should be set to provide the basis for action limits. Since stem cell products generally cannot be subject to terminal sterilization and virus removal processes, with high risk of exogenous factor contamination, microbial safety indicators (including sterility, mycoplasma, endotoxin, etc.) are tested in appropriate links during the production process.

B. Process Change

　　The change of stem cell products, can follow the basic principle of comparability study of general biological products. However, due to the complexity and heterogeneity of stem cell products, the existing pharmaceutical characterization methods may not be able to comprehensively elucidate the differences in the qualities (safety and efficacy) of products before and after the change. In the development and post-approval changes of stem cell products, more non-clinical and clinical studies may be needed.

　　For stem cell products, the comparability of product quality before and after change cannot be explained only by the detection of final products. The content of change study should cover the comparison of raw materials before and after change, process, quality attributes between process intermediates and final products, and stability, among which the comparison of quality attributes should focus on identification, biological activity, purity and impurities. The potential impact of various changes should be comprehensively identified on product safety, efficacy and quality controllability, and change strategies and study protocols should be science-based and rationally developed. Reasonable sampling points, test indicators and acceptance criteria for comparability study can be set by referring to the basic principles of ICH Q5E. The batches for comparability study before and after the change should be representative. The comprehensiveness of test items, rationality of standard setting for preset comparative study, effectiveness of test methods and objectivity of analysis of test results, should be the focus.

　　In the life cycle of the products, different production processes may be employed to produce the samples and products in different stages, such as non-registered clinical process, non-clinical process, clinical process, commercial production process and new process with post-approval changes. The differences between the processes at each stage should be analyzed in detail to study whether the product quality under each process version can be bridged. If necessary, the new non-clinical or human clinical comparison study data might be further required. In general, it is recommended that all anticipated changes be completed prior to confirmatory clinical trials.

C. Process Validation

　　The practical process validation is critical to continuously produce stem cell products with consistent quality. After process validation, all raw materials and process control should strictly follow the quality control system and standard operating procedures.

　　In process validation, multiple consecutive batches of stem cell products should be processed at commercial scale, meanwhile any impact of starting raw materials on product quality should be noticed. The maximum production capacity with the support of personnel, equipment, materials, environment, testing and other overall operation capacity, at the same time at the same stage, should be intensively concerned in commercial process validation of autologous products, as well as the support capacity of stem cells for in vitro expansion capacity, generation and differentiation efficiency, and product purity for commercial production capacity.

　　It is necessary to record in detail the changes in process performance and product quality of different batches in process validation, comprehensively confirm the robustness of production process and the consistency of product quality, verify the identification, microbial safety, purity and biological activity of samples at key production stages and evaluate the genomic stability, so as to ensure the safety of products.

　　Considering the effect of production process on the end-of-production cells, it is necessary to carry out genetic stability study and verification on the final products that can be passaged. It is recommended to focus on the investigation of growth characteristics stability, genetic stability, tumorigenicity/oncogenicity and terminal cell differentiation and dedifferentiation under the age limit.

　　Before the stem cell products are administrated into human body, transfer approach, handling methods in the hospital (such as resuscitation, washing, etc.), processing time, storage conditions and time limit of stem cell products reaching the medical institution, should also be verified. In the clinical reinfusion operation, the risks caused by the characteristics of different types of cells (such as cell inactivation, agglomeration, loss of dryness, etc.), as well as the risks of microbial contamination during clinical use, should be intensively prevent. Accuracy of dosing needs to be verified for novel delivery devices and delivery methods.

VI. Quality Study and Specification

A. Quality Study

　　Since stem cell products have the characteristics of diversity, heterogeneity, complexity, particularity and progression, the quality of stem cell products shall be comprehensively and continuously studied. It is recommended to choose representative production batches (such as non-clinical study batches, clinical trial batches and commercial production batches) and appropriate production stage samples (such as primary cells or cell seeds, cell bank, process intermediates, stock solution and finished products of preparations) for study. The content of quality study can be selected in combination with cell characteristics, covering cell characteristics analysis, physical and chemical characteristics analysis, purity and impurity analysis, safety analysis and biological activity analysis as far as possible. The advanced and orthogonal analysis techniques should be used as far as possible. These analytical methods should be confirmed by studies to ensure that the methods are applicable and reliable.

1. Cell Characterization

　　Cell Morphology: Morphological analysis may play a role in indicating the growth and differentiation status of cells. Observation on cell morphology can be performed in combination with various imaging techniques to help determine the status of cells.

　　Cell Identification: It is recommended to identify cells by various methods such as species identification and cell lineage from various dimensions such as phenotype or genotype of cells. The development of methods that can identify potentially contaminating cells and control the risk of cell cross-contamination during production are encouraged.

　　Cell Viability: Stem cell products are usually cells alive, so the viability the cells can be comprehensively evaluated by cell survival rate, viable cell number, population doubling time (PDT), and cell cycle.

　　Biomarkers: A variety of surface markers can be helpful to characterize cell types, pluripotency, lineages, terminal differentiation and/or function. Related analytical assays such as Western blot (WB), flow cytometry, and immunofluorescence are often used to analyze cell characteristics. mRNA-based markers can be used in cell characterization if effective correlation of mRNA markers and protein marker expression has been validated.

2. Physicochemical Characterization

　　For the analysis of general physical and chemical properties, it is necessary to carry out studies in combination with product type and preparation characteristics, often including appearance, color, pH value, obvious visible particles, osmolality, loading volume and other items.

3. Purity and Impurity Analysis

　　In the process of stem cell products, non-cellular impurities (such as physical and chemical impurities), cell debris or non-target cells may be introduced or produced in the production process, affecting the purity of the product and possibly bringing safety risks. Therefore, it is necessary to carry out comprehensive and standardized purity and impurity studies according to the product type and process characteristics.

　　Usually the study items for purity analysis may include: Proportion of viable cells, proportion of cell population or clades, proportion of target cells and proportion of non-target cells. After investigation, when the non-target cells have no adverse effect on the safety and efficacy of the product, it is necessary to study their composition and proportion and try to control the consistency between batches.

　　The study items for impurity analysis include process related impurities and product related impurities. Process-related impurities refer to the impurities introduced in the production process, such as residual exogenous proteins, antibiotics, induction reagents, micro-carriers, viral vectors, DNA, etc. Product-related impurities include non-target cells, products of unexpected cell expression, dead cell residues, cell debris and other possible degradation products. These impurities concerned with the safety of the products should be removed in the process, tested in the quality study and controlled qualitatively/quantitatively. It should be paid special attention to that, in the case that there may be high-risk impurity components in the product (such as ESC or iPSC residues). The methods of impurities clearance and quantitative detection for impurity residues should be established and clarified. If the impurity cannot be effectively removed, the assessment for safety and toxicity of the samples with those impurities should be conducted in animal models or other systems. The Reasonable residue limits should be set based on the results of maximum human exposure dose or in vivo safety studies to ensure the safety.

4. Safety Analysis

4.1 Biological safety:

　　The biological safety of stem cell products covers the safety concerns caused by the biological characteristics of stem cell products themselves and related to inducing changes in other cell biological characteristics in vivo conditions of recipients. The clinical indications, administration route, dose and other factors directly related to clinical treatment of relevant cells should be considered in the evaluation of biological safety of stem cells and appropriate in vivo and in vitro test models should be used to effectively evaluate the relevant biological safety. Stem cell biological safety includes tumorigenicity and oncogenicity, unexpected differentiation, and off-target editing.

　　Tumorigenicity and Oncogenicity: The risk of tumor formation and tumorigenicity of stem cell products, especially high-generation, or stem cell products that have been complex treated and genetically modified in vitro should be in the consideration from CMC perspective. Based on the three-germ layer differentiation potential of pluripotent stem cells and their potential teratogenic property, the detection of tumorigenicity and oncogenicity of final products from derived from ESCs and iPSCs should be intensively monitored. Soft agar colony formation assay and telomerase activity assay can help to detect the tumorigenicity of the product in vitro to some extent.

　　Unexpected Differentiation: Stem cells may differentiate into other cells instead of target cells in manipulation in vitro. It is recommended to develop specific detection technologies (such as high-throughput sequencing) to study, evaluate and monitor the possibility and impact of unexpected differentiation of stem cell products, which can be specifically analyzed in combination with the efficiency of target cell differentiation.

　　Off-Target Editing: The application of genome editing technology may bring different degrees of risk of off-target genome editing, with chromosomal instability and off-target editing (such as DNA insertion or deletion). For products that perform genome editing on stem cells, off-target editing in genetically modified cells should be analyzed and studied, and multiple orthogonal methods including unbiased whole genome analysis (e.g., in silico, biochemical, cell analysis methods) are recommended to identify potential off-target sites.

4.2 Microbiological safety:

　　Since stem cell products would not go through sterilization process and virus removal steps, microbial safety risks should be controlled in raw materials and production process. For details, refer to pharmacopoeia requirements and other control strategies for related drugs. Microbiological safety specifically refers to that stem cell products shall be free from contamination by various microorganisms (bacteria, fungi, mycoplasma and viruses, etc.) and microbial metabolites/derivatives (such as bacterial endotoxin). Viral contamination, including species-specific viruses (such as human-derived/animal-derived viruses), endogenous and exogenous retroviruses and other non-specific viruses, should be detected at appropriate stages according to the product characteristics. The risk of viral contamination in the product should be assessed comprehensively in the whole production process. The approaches to detect retroviruses and non-specific viral agents can refer to the relevant requirements in the General Requirements "Preparation and Quality Control of Animal Cell Substrates for Production and Testing of Biologics" of Chinese Pharmacopoeia Volume III. For stem cell products obtained with replication deficient and other viral vectors, the risk of reverse mutation of viral vectors shall be fully considered during product design and quality study. The approaches to detect replication competent virus (RCV), please refer to relevant guidelines.

5. Biological Activity Analysis

　　Stem cell products are live cell drugs, with their biological effects by multi-target, multi-pathway. Different type of stem cell products has different biological functions. Therefore, there is a need to develop quantitative biological activity/function assays that are representative of the product’s mechanism of action based on the product's clinically relevant therapeutic activity or expected biological effects. Multi-activity component products should be identified and measured separately.

　　Sometimes, it is also necessary to develop alternative assays for stem cell biological activity, such as physicochemical analytical assays performed outside of living systems, to provide extensive product characterization data by assessing the immunochemical, biochemical and/or molecular attributes of the product. For the adopted analytical method, alternative method and activity correlation study should be carried out. It should be proven that this assay can distinguish active products from inactive or degraded products, and sufficient control study and methodological validation should be carried out. Some stem cell products have complicate and/or incompletely clear mechanism of action or have a broad spectrum of biological activities. The characterization of cell biological function must be combined with the comprehensive evaluation of in vitro biological effects and in vivo animal models.

　　Stem cell products technology has a rapid progress and cognitive update, so biological activity analysis needs to keep up with the progress in combination with product characteristics. For example, up to date, the biological activities of mesenchymal stem cells (MSCs) mainly include induced differentiation function, immune regulation function and tissue regeneration function, of which the induced differentiation function includes the differentiation function detection of osteogenic, adipogenic and chondrogenic cells, while the immune regulation function can be characterized by the detection of immune regulation factors. For another example, the biological activity of ESCs/iPSCs derived cells is based on the detection of special biological functions of differentiated cells, or the detection of specific genes and proteins related to the function of differentiated cells. In addition, the detection of stem cell biological activity may also involve the detection of cell directional differentiation efficiency, the formation of cell and extracellular matrix/structure, cell interaction (such as immune activation or inhibition), cell migration and differentiation or self-renewal potential evaluation, and so on.

B. Specification

　　The establishment of quality specification mainly includes the following steps: Determine the content of quality study, conduct methodology study, determine the items and limits of quality specification and establish and revise the quality specification. Validated analytical method should be adopted in the specification of stem cell products to evaluate the product’s identification, purity, sterility and activity and should reveal the quality characteristics of stem cell products.

　　The criteria and limits of quality specification are determined in combination with quality study and product characteristics. The limit should be set to ensure the safety, efficacy and batches consistency of the product. The quality criteria of stem cell products generally include: cell identification, cell activity (cell survival rate, number of viable cells, number of functional cells, population doubling time), purity (e.g., proportion of target cells), biological activity (e.g., differentiation efficiency, quantitative/semi-quantitative functional assays, markers), product- and process-related impurities, tumorigenicity/oncogenicity (if applicable), unexpected differentiation, microbial safety (sterility, endotoxin, mycoplasma, endogenous and exogenous viruses), general tests (appearance, pH, visible particles, osmolality, loading volume), etc. If cell products have received gene modification, the percentage of target cells with gene modification, the average vector or plasmid copy number of single cell, the function of target gene and its expression product, off-target editing and the residual amount of vector for transduction/transfection should be controlled for each batch of final products. For cells transduced with a replication-deficient virus, the absence of a replicating vector (RCV) should be demonstrated. For stem cell products that need to be re-operated before administration (such as container conversion, physical state conversion, combination with other structural materials, filtration and cleaning, etc.) after transportation to the hospital, it is necessary to carry out clinical-use related studies by simulating actual operation, clarify the operation steps and precautions before administration according to the study results, and provide the hospital with full product information and operation training data, including the review of operation steps and label verification. Checking procedures before use must include, at a minimum, tests for appearance, color, identification, and package integrity.

　　Methodological study includes the selection of methods and the validation of methods. Special considerations are required in methodology study of stem cell products, for example, (1) when more advanced methods and technical improvements are used in cell substrate characterization and testing, comprehensive comparative validation of old and new methods and testing bridging studies should be performed to demonstrate that the specificity, sensitivity, and precision of the new method are at least equivalent to those of the existing method. (2) Validation of analytical methods related to biomarkers should be carried out in a standardized manner, with special attention to the design and validation of positive controls. (3) It is necessary to adopt multiple orthogonal analytical detection methods as far as possible to carry out biological activity determination study and complete methodological validation. Potential non-additive effects between active components, such as interference effects or synergistic effects, should also be considered in method studies. (4) Stem cell products may contain heterogeneous cell populations. It is necessary to quantitatively detect target cells and non-target cell populations related to biological activity using as sensitive as possible. Special attention should be paid to the setting and verification of controls for method research. (5) In principle, release testing of final stem cell products should be conducted according to the sterility test and mycoplasma test of biological products in the current edition of pharmacopoeia, and methodology validation should be conducted when rapid method is used for release when necessary. For rapid method validation, relevant guidelines should be referred to, and representative contaminated positive controls should be fully considered. When it is not fully verified that the new method can completely replace the traditional compendial method, it is recommended to carry out parallel testing of the traditional compendial method while using the new test method for release testing, and develop a handling plan for the unexpected results that may occur after the test results. The rapid method should be compared with the pharmacopoeia method step by step to prove that the detection capability is not lower than the pharmacopoeia method. (6) For genetically modified stem cell products, if exogenous genetic material is removed from the final product, it is necessary to develop relevant highly sensitive detection methods for confirmation.

　　Reference item/control article may be used in analytical test methods, and the development and establishment of cell reference item is encouraged for the quality control of stem cell products. The reference item/control article used for analysis shall be representative and traceable. The validated analytical test method shall be used for sufficient characterization and qualification. It is necessary to complete the calibration of reference item/control article (content calibration and activity calibration), carry out stability studies on the reference substance used at each stage of product development, and determine the retest period and shelf life.

VII. Stability Study

　　For the stability study of stem cell products, refer to the technical requirements for stability study of biological products, such as ICH Q5C and Technical Guidelines for Stability Study of Biological Products.

　　The purpose of the stability study is to support the storage, transportation and use of stem cell products. The study generally includes stress test (temperature, light, mechanical force, etc.), accelerated test, long-term test, transportation test and clinical in-use stability test. Test samples generally include representative stock solutions (if any), finished products and process intermediates requiring temporary or staged storage. The production, use and quality (such as total cell density and volume range) of samples for research should be representative of the actual situation. The differences between fresh cells and cryopreserved cells should be fully considered for the test conditions, the special requirements for the storage, packaging, transportation, clinical compatibility and actual administration of stem cell products should be considered, and the impact of the cumulative storage time of samples in each link on the stability of the final product should be considered. The test items should be able to fully reflect the influences of the study conditions on the quality, such as cell characteristics such as cell survival rate and number of viable cells, biological activity, cell purity, physicochemical characteristics, microbial safety indicators and content of key excipients.

　　The stability data at the clinical stage under application should be able to support the implementation of clinical trials, that is, the stability study period should at least cover the requirements of the clinical trials carried out, and prove that the shelf life of the product from release to patient administration is reasonable. When applying for marketing, it is necessary to provide the stability data of multiple representative batches to support and determine the storage conditions, service conditions and shelf life. In addition, it is necessary to make clear the sensitive conditions, cell state or the change of quality over time of product.

VIII. Packaging and Container Sealing System

　　The objects of suitability assessment of the packaging and container sealing system refer to the package containers and sealing system in direct contact with products. Appropriate internal packaging materials (freezing tubes, vials, flexible bags, etc.) should be selected in combination with the route of administration (intravenous administration, local administration, ophthalmic preparations, etc.) and the nature of preparations (fresh cells, frozen preparations, etc.).The functionality and compatibility of the immediate packaging materials and sealed containers should be studied. Functional studies, such as airtightness and low temperature resistance, can be carried out by referring to relevant technical guidelines.

IX. Glossary

Adult stem cells (ASCs):

　　Undifferentiated cells derived from developing embryonic tissues, adult tissues (e.g., tooth-derived, fat-derived, etc.), or birth associated adnexal tissues (e.g., umbilical cord, placenta) can self-renew and have the potential to differentiate into one or more terminally functioning cells, such as, mesenchymal stem cell (MSC), hematopoietic stem cell (HSC), neural stem cell (NSC), and keratinocyte stem cell (KSC).

Human Embryonic stem cells (ESCs):

　　Initial cells derived from the differentiation of human preimplantation embryos should not be cultured in vitro for more than 14 days from fertilization or nuclear transfer, can be self-renewing without restriction in vitro, and have the potential to differentiate into three-germ layer cells.

Induced pluripotent stem cells (iPSCs):

　　A kind of stem cell obtained by reprogramming human cells with unlimited self-renewal ability and differentiation potential into three-germ layer cells, which has multi-functionality similar to human embryonic stem cells.

Transdifferentiation:

The process of directly transforming somatic cells or adult stem cells into other somatic cells or stem cells of different germ layers or different lineages without pluripotent stem cell state through genetic modification, chemical induction and other methods.

Teratoma:

　　A benign tumor containing differentiated tissue and cells of three germ layers. The scientific community often verifies that the teratoma produced contains three germ layer cells by injecting stem cells into immunocompromised mice. It can be used to determine whether a human embryonic stem cell line or a human induced pluripotent stem cell line has been established.

Feeder layer cell:

　　It can be used for supporting the growth and expansion of other cells through cell - cell interaction or secretion of certain nutrients.

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