National Medical Products Administration, Center for Drug Evaluation

　　May 2022

I. Introduction

　　In recent years, technologies like cell therapy and gene editing are developing rapidly, and the relevant medicinal products are constantly evolving, which provide new treatment philosophies and methods for serious and refractory diseases. The increasing clinical needs have facilitated the application and updating of new technologies for genetic manipulation.

　　Ex vivo, the gene modification system constructed by genetic engineering technology can effectively transfer synthesized genome sequence into the specific target cells, which will be modified to change gene expression or regulate the biological characteristics. Nowadays, lentiviral vectors, γ-retroviral vectors, etc. are commonly used to introduce Chimeric Antigen Receptor (CAR) gene into T cells to achieve targeted killing of tumors by CAR-T cells. Free Episomal Vectors, Sendai virus vectors, etc. can be used to introduce transcription factors into cells, to obtain induced pluripotent stem cells by reprogramming and provide starting raw materials for the production of their derived cell products. In the future, more diverse vector designs are expected to be applicable to different types of products.

　　There are various types of gene modification systems. The differences in vector design, manufacture process and quality control directly affect the safety and effectiveness of the final product. In addition, the source may be different and the differences may exist in the quality management system. In order to ensure the quality of gene modification systems meet the requirements of clinical application, sufficient quality study is required. Therefore, it is necessary to define the technical requirements for pharmaceutical study on different types of gene modification systems.

　　Based on the current scientific knowledge, the guideline proposes the recommended technical requirements for the application for NDA of gene modification systems for ex vivo use, which is intended to provide guidance for the R&D units and serve as an important reference for the evaluation by the regulatory authorities. This guideline is not mandatory. If there are other alternative or applicable study methods, or not applicable contents with this guideline, the applicants/holders can provide the supporting reasons and basis for the relevant alternative study. With the development of technology, evolving of cognition and accumulation of experience, the contents of the guideline will be gradually revised and improved.

II. Scope

　　In this guideline, gene modification system refers to the modification system introducing exogenous genes ex vivo into cells and add, replace, compensate, block and correct specific genes so as to obtain cell therapy products or seed cells for the production of cell therapy products. Possible mechanisms include expression of functional target genes in cells, or change of specific gene sequence by nucleotide editing methods such as gene knockout, repairing and insertion.

　　At present, gene modification systems include viral vectors such as lentiviruses, γ-retroviruses, adenoviruses, adeno-associated viruses, and Sendai viruses, as well as non-viral gene modification systems such as DNA, RNA, proteins, and protein-RNA complexes. The Guideline discuss two types of gene modification systems, viral vectors and non-viral vectors. With the continuous change of technologies in this field, new gene modification systems may also be applied. If applicable, the pharmaceutical study may also refer to this guideline.

III. General Principles

　　The molecular design, process consistency and quality control of gene modification system can directly affect the safety and efficacy of final cell products, thus making them the focus of the Chemistry, Manufacturing and Controls (CMC) study. The CMC study to be conducted may include gene modification system design, manufacturing process development, quality study and control, stability study, etc. In principle, the whole manufacturing process of gene modification system should meet the requirements of Good Manufacturing Practice for Drugs (GMP). Specific requirements can be applied to corresponding conditions, which will be evaluated on a case-by-case basis.

　　Although the gene modification system of ex vivo gene products shared some similarity in quality risks to those of in vivo gene therapy products, they are in principle different. Ex vivo genetically modified cells may undergo ex vivo cell culture, wash and cleaning steps, and final cell product release testing before being applied in human use. Compared with in vivo gene therapy products, the quality attributes of gene modification systems can be better controlled before transfusion, and some related impurity can be controlled before product verified testing. The design, quality study and control of gene modification system for ex vivo use are recommended to be based on their characteristics.

　　(I) General Requirements for Different Development Stages

　　Like the development of other drugs, the CMC study of gene modification systems also follows a phase-appropriate manner and is adjusted with the advancement of the non-clinical and different clinical study stages of the final cell products. The sponsor is encouraged to develop the study plan and strategy in advance and carry out relevant studies based on the concept of “quality by design”. With the progression of development stages, the sponsor can gradually optimize the manufacturing process and improve quality control of the gene modification systems.

　　At IND stage, sponsors shall identify and control the risks associated with gene modification system, describe the molecular design, complete the production and characterization of the seed bank (if applicable), evaluate the rationale of raw material selection and their safety for manufacturing. Sponsors are recommended to establish a relative consistent manufacturing process through process development study, and specification of the product through quality attributes study to assure the safety of gene editing system and its modified cells in clinical use.

　　With deeper understanding of the critical process and quality attributes, the commercial manufacturing process for gene modification system shall be established at BLA stage after further optimization, comprehensive characterization and validation work.

　　If the process of the gene modification system changes during the clinical stages, the comparability study of the gene modification system and the corresponding cell product shall be completed; it is recommended to complete the major changes and confirm the process before the pivotal clinical trial. It is recommended to develop reasonable quality control strategy and specify Critical Quality Attributes (CQA) based on comprehensive and in-depth quality study and accumulated data from multiple batches of manufacturing. Appropriate analytical method shall be developed and comprehensively validated. Sponsors are recommended to study gene modification system-related as well as process-related impurities and develop corresponding risk control strategy. Reasonable storage condition and shelf life of the final product shall be determined based on stability study of the gene modification system and compatibility study of container closure systems.

　　(II) General Requirements for Gene Modification Systems from Different Sources

　　Gene modification systems can be self-produced, contract manufactured, or directly purchased. Regardless of the source, all gene modification systems shall follow the same technical guideline and quality control principles. The CMC study can be carried out following this Guideline.

　　Marketing authorization holders of final cell products shall bear the main responsibility for the quality of gene modification systems. They shall control corresponding quality risks by strengthening internal quality control or managing the contracted manufacturers of gene modification systems through quality agreements and regular audits. If there are changes of gene modification system, the risk shall be assessed in a timely manner, corresponding comparability studies shall be conducted, and in some cases, nonclinical or/and clinical bridging studies may also be required.

IV. Risk Assessment and Control

　　(I) Overall Risk Identification and Control Strategy

　　The risks involved in gene modification system mainly include the reverse mutation of viral vector, oncogenicity or pathogenicity caused by the vector integration in the genome, off-target risks, contamination of adventitious agents and residual impurities, etc.

　　Pharmaceutical study can carry out the analysis of risk factors from multiple aspects such as the design, manufacture process and quality control of modification system, identify and determine the risk factors related to product quality and safety, determine the data ranges and focus of risk assessment required during research and development, and develop risk prevention and control and handling measures. At the same time, risks shall be considered comprehensively in combination with cell type, dose, route of administration, population, mechanism of action, in vivo distribution, in vivo duration of action and other aspects.

　　In terms of the design of gene modification system, typical risk factors may include: the use of risk elements, sequence recombination possibly caused by homologous sequences, reverse mutation of viral vectors after complementation of corresponding wild-type or auxiliary viruses, incubation/reactivation and/or mobilization of vectors in cells, degree of chromosomal integration of vectors in cells and preference of integration sites, etc. In terms of manufacture process, relevant risk factors may include use of high-risk raw materials, contamination of adventitious agents during manufacture, storage and quality control of intermediates, introduction and generation of harmful impurities, and stability of gene sequence of modification system. In terms of quality control, relevant risk factors may include: the applicability of test methods, the rationality of specification, etc.

　　In recent years, enzyme-based gene editing systems have been gradually applied to gene modification of cell therapy products, and commonly used systems include Transcription Activator-Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Protein (Cas), etc. The risks of clinical use of such system mainly include the self-toxicity and immunogenicity of genetic tool enzyme, genotoxicity caused by on-target and off-target during gene editing, and impurity residues, etc.

　　Therefore, risk assessment and control shall be performed according to multiple factors, considering the characteristics of different types of gene modification systems and in combination with the characteristics of the final cell products. For example, according to the risk assessment, it is recommended to rationally design the structure and sequence of the modification system, avoid the use of high-risk elements such as carcinogenic elements, carry out relevant tests and reduce the possibility of homologous recombination and reverse mutation of viral vector as far as possible; carry out quality control on high-risk raw materials for production, set the test items and acceptance criteria for in-process control in the appropriate manufacture stages; carry out the study on the safety-related risk factors such as gene sequence stability according to the mechanism of action of the gene modification system. Track, analyze and update the modification system throughout the lifecycle of cell final products to collect data to further determine its risk characteristics and develop control strategy.

　　(II) Risks under Different Situations of Manufacturing

　　The use of gene modification system in the production process of cell therapy products may be different. The current study may include two use situations: cell products directly manufactured after ex vivo gene modification (Case 1) and cell products manufactured after ex vivo gene modification followed by constructing cell lines/banks construction (Case 2). Since the risks of corresponding gene modification system are different in the two use situations, there may be differences in the requirements for pharmaceutical study, and it is necessary to be analyzed on a case-by-case basis.

　　For Case 1, it is recommended to manufacture the gene modification system under the conditions of GMP. The gene modification system used in this case is in direct contact with the cells implanted to human body. The quality control of the raw materials used for the production of this system, the quality differences between batches, impurity levels and other study contents that may affect the safety and effectiveness of cell therapy products shall be concerned. The later part of this Guideline mainly discuss this situation.

　　For Case 2, the gene modification system is used for the establishment of cell line/bank, and the requirements for the sequence design, materials for production, manufacture process, quality, stability and primary packaging materials may be comprehensively evaluated according to specific conditions and in combination with the quality study results of cell line/bank. Due to the complexity of the use of gene modification systems (such as single use or multiple uses), the risk control strategy of modification systems can be developed in combination with the situation of manufacture and use and the study and testing of cell lines/banks. Extensive quality study and rational quality control of gene modification system is conducive to screening, establishment of good quality cell lines/banks, and subsequent production study. During the establishment of a cell line/bank, it is recommended to conduct monoclonal screening, carry out verification and passage stability study on the cell line/bank, pay attention to whether the functions of the cell line/bank meet the theoretical design and expectations, whether the safety is controllable, and carry out corresponding quality study on gene modification on the final cellular products if necessary, so as to confirm the applicability of the gene modification system.

　　(III) Change Risks

　　With the continuous updating of gene modification technology and the gradual accumulation of research experience, the R&D process is often accompanied by the upgrading of gene modification system and process optimization, so the gene modification system may be changed at each stage of R&D.

　　The change of gene modification system may significantly affect the safety and efficacy of cell products which is one of the important risks in the pharmaceutical study of cell therapy products. The sponsors shall evaluate the impact and risk of the introducing change. Conduct deep study on quality, process control and stability of gene modification system and final products (if applicable) based on risk assessment, and reasonably design the comparability study protocol.

V. Design, Manufacture and Quality Control of Gene Modification System

　　(I) Viral Vector Gene Modification System

　　1. Design and Construction of Viral Vectors

　　1.1 Target Genes and Regulatory Elements

　　Target genes are key sequences to achieve intended functions in gene modification systems, while regulatory elements are important sequences that influence transcription, translation and stability of target genes. The sponsors shall design and construct each element of gene modification systems based on safety and efficacy of final cell products and risk assessment.

　　Target Genes: Target genes in the Guideline refer to genetic materials transmitted or expressed by gene modification systems. The sponsor shall carefully select and design target gene sequences according to their roles or mechanisms of action in disease treatment, and pay attention to the origin of target genes and the process of sequence screening and optimization. The complete nucleotide and amino acid (if applicable) sequence information shall be clear. It is recommended to compare the sequences with relevant sequences in the database. During optimization of target genes, scientific evaluation and validation studies of molecular modification shall be described, such as humanized modification, knockout elements, suicide markers, and sequence modification and deletion for regulating formation of higher order structures or adapting to vector size. Meanwhile, it is recommended to evaluate potential non-specific effects according to the specific binding ability of target genes or their expression products to target molecules. Effects of target genes on stability of target cell genome can be fully considered based on sequence characteristics of target genes and the interaction between target proteins and cell genome. If target genes belong to non-human or modified sequences, immunogenicity of target genes can be evaluated considering the difference of target gene sequences between species and the immune characteristics of expression products.

　　Regulatory Elements: Regulatory elements play important roles to regulate and control transcription and expression of the genes of interest. It is recommended to pay attention to their design principles and make confirmative studies. Regulatory elements can be rationally selected and designed according to expression levels, expected effects and duration of target genes as well as target cell types. Relevant regulatory elements may include signal peptides, promoters, enhancers, terminators, insulators, 5'-untranslated regions, 3'-untranslated regions, polyadenylation signals, introns, replication initiation sites, other elements to enhance transcription and translation efficiency, and additionally introduced gene sequences. The sequence origin and selection basis of each regulatory element shall be clarified. For example, in terms of promoter design, it is recommended to analyze the safety according to target cell types, requirements of expression duration and levels of target genes, and experience of promoter applications in humans, so as to select the promoter rationally on the basis of risk assessment. In the design of regulatory elements, it is necessary to fully consider safety and effectiveness of elements, pay attention to necessity and rationality of introduction of relevant sequences, avoid the use of high-risk elements as far as possible, and if necessary, modify relevant sequences to increase safety. For the modified or optimized elements, the basis and safety considerations of sequence changes shall be clarified. In addition, it is also recommended to focus on interference of functional elements with endogenous genes in cellular genomes.

　　1.2 Viral Vectors

　　Viral vectors can usually transduce target genes to target cells stably and efficiently. For selection and design of viral vectors, expression duration and amounts of target genes, pathogenicity, integration capability, infection process, transduction efficiency, cytotoxicity, cell types, mechanisms of action, indications and administration routes of cell products may be comprehensively considered. Structural optimization strategies of viral vectors include improving viral stability, enhancing cell transduction efficiency, and broadening transmissible cell types.

　　Currently, commonly used viral vectors usually undergo deletion of genes related to virulence, pathogenicity, or replication capacity to ensure safety. During design, any pathogenicity related to wild type virus shall be reduced as far as possible, and risks of virus recombination and reverse mutation shall be minimized as far as possible. For modified viral vectors, attentions shall be paid to the source, culture history and biological characteristics of parental viruses, and materials, methods, protocols and identification for modification shall be fully studied. No additional safety risks shall be introduced when viral vectors are modified.

　　The following sections are illustrated with common viral vectors as examples.

　　1.2.1 γ-Retroviral Vectors

　　γ-retroviruses can reverse transcribe their RNA genomes into DNA copies, which are randomly integrated into mitotically active cellular genomes. At present, γ-retroviral vectors for ex vivo gene modification generally include murine leukemia viruses (MuLV), feline leukemia viruses (FeLV) and gibbon ape leukemia viruses (GaLV), etc. Design and modifications of γ-retroviral vectors mainly include improving vector safety and gene transduction efficiency.

　　γ-retroviral vectors can be packaged by transient transfection of cells with transfer plasmids that contain target genes and auxiliary plasmids. Alternatively, they can be prepared by stable virus-producing cell lines with genomically integrated transfer plasmids and auxiliary packaging elements. To improve safety of modified vectors, it is recommended to optimize the modification system, such as using γ-retroviral vectors which are deleted of non-essential elements, fully modified and safer. Risks of viral recombination and reverse mutation shall be minimized as far as possible. Optimization strategies may also include using auxiliary packaging elements with heterologous promoters and Poly A signals, splitting auxiliary packaging elements into different plasmids, and engineering long terminal repeats (LTRs) to make the ends self-inactivated (SIN).

　　In terms of gene transduction efficiency, sponsors are encouraged to optimize packaging elements of viral vectors and improve packaging efficiency, structural stability and transduction activity of viral vectors. For example, the envelope protein of γ-retroviral vectors can be replaced with other envelope proteins to improve viral stability. Adequate research and validation are required for modifications.

　　1.2.2 Lentiviral Vectors

　　Lentiviral vectors play a role by mediating integration of target genes into the cellular genome. Unlike γ-retroviral vectors which can only infect mitotically active cells, lentiviral vectors can infect both mitotically active and inactive cells. At present, lentiviral vectors for ex vivo gene modifications generally include human, non-human primate and non-primate lentiviruses. When using non-human primate and non-primate lentiviral vectors, it is recommended to focus on risks of producing recombinant chimeric viruses and/or cross-species transmission. Design and modifications of lentiviral vectors mainly include improving safety and transduction efficiency.

　　The main risks for manufacture and clinical use of lentiviral vectors include production of replication-competent lentiviruses (RCL), occurrence of in vivo recombination, and insertion of provirus DNA in or near certain genes that may cause or promote tumorigenesis and other cytopathic effects. Therefore, in terms of lentiviral vector design, it is recommended to adopt all measures that may weaken pathogenesis, including separating packaging genes in different expression plasmids (e.g., separating gag/pol and rev), reducing sequence homology between auxiliary and transfer plasmids, deleting unnecessary regulatory elements, and engineering LTRs for end self-inactivation. For transfer plasmids, it is encouraged to perform sequence optimization to improve safety, such as reducing the chance of proto-oncogene activation caused by promoter sequence insertion into target cells.

　　In order to improve gene transduction efficiency, modification strategies such as replacement of envelope proteins and improvement of site-directed integration efficiency can be considered. For example, human immunodeficiency virus (HIV)-derived lentiviral vectors can be prepared by replacing the HIV envelope gene sequence with a gene encoding a heterologous viral envelope protein (e.g., VSV-G) to enhance vector tropism and stability. Through modification of integrase and LTR sequences, site-directed integration of lentiviral vectors can be improved. When modifying vectors, it is recommended to consider possible risks introduced by changes in stability of viral vectors and preference of integration sites.

　　1.2.3 Sendai Viral Vectors

　　Sendai viral vectors are single-stranded antisense RNA viral vectors that express target genes in the cytoplasm and usually do not integrate into the cellular genome. They have been reported to be applied during cell reprogramming to establish induced pluripotent stem cell lines (iPSCs). During design and construction, while stably expressing the target gene, it may be considered to delete or modify viral assembly-related proteins, such as nucleocapsid structural proteins and matrix proteins involved in viral assembly, to prevent production of replication-competent virus. To ensure effective control of residual Sendai viral vectors in iPSCs, replication-related elements can be modified, such as construction of Sendai viral vectors with temperature-dependent replication capabilities by mutating RNA polymerases. Meanwhile, relevant methods need to be established to detect and verify whether Sendai viral vectors remain in iPSC clones to control risks.

　　Other viral vectors may include adenoviruses, adeno-associated viruses, etc. Their molecular design and construction can be performed based on specific viral structure, target gene sequence characteristics, safety of packaging sequences and other comprehensive considerations. Refer to the above for basic principles.

　　2. Materials for Production

　　2.1 Plasmid

　　For viral vectors prepared by transient co-transfection of multiple plasmids, corresponding studies on plasmid design and construction, production process, quality control and stability need to be carried out based on risk assessments and vector characteristics, etc.

　　Design and construction: Based on the research, select a reasonable plasmid backbone, replication origin, promoter and selective markers and other component elements, remove non-essential components (especially sequences with high risk of carcinogenesis, etc.), and completely confirm the nucleotide sequence of the plasmid.β-lactam antibiotic resistance gene shall generally be avoided, and it is recommended to prevent the insertion of resistance gene sequence into the viral vector genome to the greatest extent, and encourage using non-resistance gene for screening. When additional transcription or translation enhancing elements are used, their functionality and safety shall be fully evaluated and, if necessary, modified for safety accordingly, such as the Woodchuck hepatitis virus posttranscriptional regulatory element(WPRE). It is recommended to split different packaging elements into multiple plasmids for expression whenever possible to reduce the probability of homologous recombination.

　　Manufacturing Process: Based on the process study, a stable scale up production process for plasmid shall be determined by exploring and optimizing the key process parameters. The manufacturing process shall have a clear detailed description of the scale, process flow, process steps and process control strategy. In manufacturing process, human-and animal-derived materials and β-lactam antibiotics shall be avoided as much as possible, and if other types of antibiotics are used, the residual amount of antibiotics shall be controlled and safety assessed. Based on the research and risk assessment, carry out plasmid process validation or confirmation studies, such as process control confirmation, intermediate product storage stability studies, multi-batch quality analysis and impurity removal studies, etc.

　　Quality Control: It is necessary to establish reasonable specifications and conduct release testing. The quality control items generally include: pH, appearance, identification (restriction enzyme analysis and sequencing), plasmid concentration/content, purity and impurities (A260/A280, supercoiled plasmid ratio, residual host bacterial DNA, residual host bacterial RNA, residual host protein, residual antibiotic (if applicable)), sterility and bacterial endotoxin.

　　Stability Study: Select reasonable and sensitive items (such as supercoiled plasmid ratio, etc.) for stability monitoring, and establish reasonable storage conditions and shelf life according to the study results.

　　2.2 Bacterial Seed Lot

　　Bacterial seed lots in the Guideline refer to the seed banks established by transformation of packaging plasmids into host bacteria, monoclonal screening, culture and passage. For the selection of host bacteria, it is necessary to fully consider the source, genotype, phenotype, previous use experience, production needs and other factors of host bacteria. If new strains are used, it is necessary to assess the additional risks may be introduced.

　　Establishment of bacterial seed lots: Establish bacterial seed lots at all levels according to the study, and specify the manufacture scale, amplification conditions and storage conditions. The study shall focus on the screening of target clones, source of materials used to generate the seed lot and production process so as to assess and analyze safety risks.

　　Quality control of bacterial seed lots: Establish appropriate release test items, criteria and test methods. Test items generally include bacterial morphological identification, staining microscopy, biochemical characteristics analysis, resistance test, plasmid restriction profile analysis, and accuracy analysis of target gene sequence and other elements. Test methods need to be validated and/or verified. Quality control shall ensure that there is no contamination by other bacteria, fungi and phages, and ensure the accuracy of target gene sequence and other element sequences.

　　Study on the stability of bacterial seed lot: The passage stability generally includes plasmid size, accuracy of plasmid sequence, plasmid restriction profile analysis, plasmid retention rate and copy number. The limited passage number of seed lot shall be clarified according to the study results. For the stability of storage, it is recommended tofocus on the survival rate of seed lot under storage conditions and within a period of time.

　　2.3 Production/Packaging Cell Bank

　　The cell matrix involved in the manufacture of viral vectors includes the cell matrix for packaging viral vectors and the stable cell line for stable production of viral vectors. When selecting a cell matrix, the feasibility of viral vector packaging and manufacture needs to be considered, in combination with the cell source (including species origin), growth characteristics and vector manufacture capacity, as well as cell characteristics that may affect the safety of the final product, etc., to select a suitable cell matrix. In the risk assessment, it is necessary to fully consider whether there are endogenous viral particles and carcinogenic sequences in the cell matrix. For newly established cell matrices or cell matrices with corresponding risks (e.g., cells derived from tumor cells), cells shall be assessed for the tumorigenicity and carcinogen icity where applicable. In some cases, modification of the cell matrix is required (e.g., insertion of specific viral sequence to allow viral replication or packaging), the study shall pay attention to the genetic, epigenetic and growth characteristics of modified cells and the manufacture of viral vectors. Cell banks are established after cell matrix selection and/or modification to ensure stability and consistency of production. This can be done by referring to the Chinese Pharmacopoeia, ICH Q5A, ICH Q5D, etc. The test items shall be determined based on risk assessment, including at least identification, sterility, mycoplasma, spiroplasma (insect cells) as well as endogenous and exogenous viral agents, species-specific viruses. Carry out the study on passage stability of cell bank, including cell growth stability, genetic stability, stability of viral vector packaging capacity, etc. It is recommended to include the end-of-production cells of viral vectors or control cells for parallel production in the study and to determines the limitation of cell passage number suitable for the manufacture of viral vectors.

　　Cell bank for packaging viral vectors: Cells in the cell bank are amplified, transfected with plasmids to synthesize viral vector gene sequences, express vector proteins and finally form viral vector particles. For example, HEK293T cells are commonly used for lentiviral vector packaging at present, and the DNA fragment between plasmid LTRs transferred into this cell is transcribed into RNA, which is packaged to form viral vector particles together with proteins expressed by helper plasmids. It is important to note that when viral vectors and non-vector DNA sequences (such as plasmid DNA, auxiliary virus sequences, host cell DNA, etc.) are co-packaged, non-vector DNA sequences may recombine homologously with viral vectors, or their residues may cause carcinogenic risk. It is recommended to carry out risk analysis and study to evaluate the rationality of the selection of cellular matrix. For example, when lentiviral vectors are packaged using cell matrices containing risk elements such as adenovirus E1 and SV40LT antigens, attention shall be paid to the residual amount of adenovirus E1 and SV40LT antigen sequences in the vectors.

　　Cell bank that can stably produce viral vectors: The cell matrix can stably express or produce proteins or components for virus packaging after gene modification, screening and library construction, and complete virus packaging and manufacture. For example, mouse PG13 and HEK293-Phoenix cells are commonly used for the manufacture of γ-retroviral vectors, and gag-pol, envelope protein, and target gene need to be stably expressed in cells for production. In the construction process, it is recommended to focus on the packaging efficiency and the quality of the viral vector produced, and to reduce the safety risks such as contamination by endogenous and exogenous agents and replication competent virus generation. Stable viral vector-producing cell lines need to be genetically modified and obtained through monoclonal screening. Cell banks should be established with the obtained monoclonal cell lines, and comprehensive testing is required. At the same time, the passaging stability of banked cell should be carried out, focusing on the stability of the inserted gene fragments in different generations of the stable viral vector-producing cells, as well as the yield and quality of the viral vector.

　　2.4 Virus Seed Lot

　　Where viral vectors or auxiliary viruses are prepared by virus seeds, virus seed lots shall be established. The source and historical culture of virus seeds should be clear and definite, and the risk can be controlled. For virus seeds with unclear culture history, risk of other virus contamination, or unconfirmed monoclonality of the viral strain, it is not recommended to use them. If it is necessary to use them, multiple rounds of plaque purification, limited dilution purification, or DNA/RNA rescue can be performed during the construction process to ensure the purity and monoclonality of the viral strain.

　　The information about the establishment process of seed lot, generation, storage and maintenance should be clear and definite; if any, describe the human-derived/animal-derived materials used in the preparation process of seed lot and perform safety evaluation.

　　The virus seed lot should be adequately tested, and the recommended testing items include: sterility, mycoplasma and adventitious viral agents, identification and sequencing of viral vector and target genes, virus titer or concentration, biological activity, impurities, sensitivity to antiviral drugs (if applicable), reverse mutation (if applicable), etc. It is recommended to conduct whole gene sequencing on virus seed lots and conduct comparative analysis on sequencing results and expected sequences, with all the differences assessed if so. For the viral vector with longer sequence, the maximum sequence analysis is recommended and the sequences analyzed are recommended to include the gene insertions, flanking region and modified regions or regions that may be susceptible to recombination.

　　For virus seed lot, it is necessary to carry out comprehensive passage stability study. The study process shall be able to represent or simulate the actual manufacture process. The study should focus on the genetic stability and production stability of virus seed lot. Based on the research, the limited passage number of the virus seed lot should be established.

　　If auxiliary virus is used for the preparation of viral vector, sufficient studies shall be conducted to explain the necessity and selection basis for the auxiliary virus, and illustrate the safety of auxiliary virus according to the scientific knowledge and production experience. The design and construction of auxiliary virus, bank construction, production, preparation and testing are recommended to refer to the general requirements of virus seed lots mentioned above.

　　2.5 Other Materials for Production

　　Other materials include raw materials (such as reagents and culture media etc.), consumables and culture containers. In combination with process study, select appropriate raw materials for production, set reasonable quality specifications for raw materials, conduct strict supplier audit, and clarify the source, components, functions, use stages and standards. The ingredients of human or animal origin shall be avoided as much as possible during the manufacture process. If it is necessary, the risk assessment and study related to the safety of adventitious agents can be carried out by referring to the relevant provisions of Chinese Pharmacopoeia and/or ICH Q5A guidelines. The raw materials requiring special attentions include: human or animal derived materials used for cell culture (such as bovine serum, digestive enzymes), plasmid transfection reagents (such as polyethyleneimine, cationic ester, etc.), nuclease, etc. For the products with higher risk such as human serum albumin used for the manufacture or storage of viral vectors, it is recommended to select the product approved by regulatory authorities and conforming to relevant national technical requirements and management specifications as far as possible. For consumables and culture containers, it is recommended to conduct analysis and study to evaluate their applicability and minimize the safety risks during the manufacture of viral vectors.

　　3. Manufacture Process

　　The manufacture process of viral vectors refers to the whole process from the recovery and expansion of cells for production, the harvest of viral vectors to the filling and storage (if any) of viral vectors. For the viral vector that directly used for ex vivo gene modification, since the quality of viral vector can directly affect the quality of final products, the study on manufacture process shall be more sufficient. The manufacture process shall be developed based on a full understanding of the overall process and the accumulated experience in the quality of viral vectors, standardized process operation steps, process control parameters and abandonment criteria shall be established, and key process parameters shall be specified. The manufacture process shall be appropriate to ensure that the cell product meets the requirements of the quality target product profile corresponding to the stage of development. In addition, measures shall be taken to avoid confusion, contamination and cross-contamination of viral vectors in the whole process of manufacture, storage and transportation.

　　3.1 Manufacture scale and batch definition

　　There are great differences in the cell types, growth characteristics, viral vector yield and stability for the production of different types of viral vectors, and the maturity of manufacture process and the dosage of viral vectors are also different. It is recommended to reasonably determine the manufacture scale of viral vectors in combination with the characteristics of cells to be genetically modified, the process of viral vectors and the clinical needs. The manufacture scale of viral vectors shall be adapted to the developmental phase of the products (clinical trials, commercial manufacture).The process may include cell culture for production, plasmid transfection or virus infection, harvest, purification and other steps. The upstream and downstream process scale in the manufacture process shall be matched reasonably. For smaller scales, it is recommended to focus on the consistency of quality between different batches.

　　The batch definition and numbering rules are developed based on the characteristics of the viral vector process. If necessary, different process steps in the manufacture process may be clarified, and special attention shall be paid to the batch definition and numbering rules when there are sub-batches or combined batches. Ensure traceability of viral vector lots.

　　3.2 Process study and development

　　There are multiple types of processes used to manufacture viral vectors, including manufacture by transient transfection of plasmid DNA into packaging cell matrices, manufacture by stable cell lines for production, or manufacture by infection of cell matrices with viral seeds.

　　It is encouraged to carry out process study in combination with new concepts of quality by design and overall process control, as well as general requirements for relevant risk control. With the accumulation of production experience and further understanding of quality attributes, the process will be continuously optimized to transforming from the laboratory process to the commercial-scale process. The manufacture process can be generally divided into two steps: upstream and downstream, that is, upstream viral vector cell culture and harvest step and downstream purification step. The type of cells for production, cell culture conditions, conditions of transfection or infection, harvest time, purification steps and hold-time conditions during the manufacture process will affect the packaging efficiency and quality of viral vectors. In the study, it is necessary to test, verify or validate the process steps, key process parameters and their control range and establish the corresponding in-process control criteria. For example, Replication competent virus (RCV) is one of the safety risks in the process control. The risk assessment shall be conducted based on the types of viral vectors and process characteristics. Reasonable testing points (such as viral vector harvest bulk, end-of-production cells or purified viral vector DS, etc.) shall be selected in the process and shall use validated methods. In addition, based on risks and in combination with the tolerance of viruses to physical and chemical conditions, if necessary, corresponding virus removal/inactivation steps shall be established and fully validated based on the cell types for production, use of auxiliary viruses and purification process characteristics.

　　In the following, the two manufacture processes, γ-retroviral vector by stable virus production and lentiviral vector by plasmid transfection are introduced respectively. Other viral vectors and other manufacture methods, if applicable, can also be referred to.

　　3.2.1 Study on manufacture process of stable virus production

　　(1) Manufacture

　　In the case of γ-retroviral vectors, they are secreted into the culture medium by stable virus-producing cells during manufacture and can be purified by clarification filtration and other steps to obtain viral vectors. It is recommended to establish rational process steps and parameters based on the structural characteristics, packaging mechanism, stability, etc. of viral vectors, and pay attention to cell culture volume, seeding density, culture conditions, harvest time, etc. during the manufacture process. For example, it has been reported that due to weak stability of some envelope proteins of γ-retroviral vectors under the usual cell culture conditions (37℃), studies are recommended to establish appropriate virus manufacture and harvest strategies to ensure the viability and yield of the viral vector.

　　(2) Purification

　　According to the structural characteristics of γ-retroviral vector, the types of host cells, and the components of impurity residues, a reasonable purification process shall be designed in order to improve the purity of viral vector and reduce the safety risk of impurity residues.

　　For example, the envelope proteins of some viral vectors may be relatively fragile to the chromatography process (such as shear force). Therefore, the development of advanced purification processes is encouraged to improve the purity of viral vectors as much as possible and realize the large-scale purification of viral vectors to meet the structural stability and functional activity requirements.

　　3.2.2 Study on plasmid transfection process

　　(1) Packaging and Manufacture

　　In the case of lentiviral vectors, the virus packaging cells used for plasmid transfection are usually cultured in adherent or suspension mode. It is recommended to select the cell culture mode according to the scale, manufacture process design and quality study for commercial manufacturing. The study of virus packaging process includes the optimization of parameters such as plasmid concentration, plasmid ratio, transfection reagent and concentration, induction reagent concentration (if any), transfection time, cell density, cell culture medium components, cell culture environment (temperature, pH, dissolved oxygen), harvest time, and harvest times. Based on the study, confirm the process steps, key process parameters and their control range, and establish the corresponding in-process control standards. For example, cell viability and bioburden are regularly tested during cell culture, vector titer, mycoplasma and exogenous virus tests are performed, and replication competent lentivirus (RCL) tests are performed. If the vector harvest solution needs to be stored, corresponding study shall be conducted to confirm the storage conditions and storage method. For adventitious agents, it is recommended to improve the sensitivity of the testing method as much as possible and carry out the testing at an appropriate process step (such as the stage of adventitious agents enrichment).

　　(2) Purification

　　At present, during the purification of lentiviral vectors, the large nucleic acid impurity segments on the surface of viral vector are removed by endonuclease first; then impurities are removed through clarification, filtration and ion chromatography or size-exclusion chromatography; finally, produce viral vector through formulation, filtration and filling. According to the study data, the purification process can be appropriately adjusted and optimized, and various purification process steps are determined to achieve the purpose of removing impurities and purifying viruses. It is recommended to study the nuclease concentration, purification method, buffer selection, dynamic loading capacity, flow rate, yield, viral vector storage conditions, filling process parameters, etc. and test nuclease residue, BSA residue, risky component residue (such as residual adenovirus E1 and SV40LT antigen sequences), plasmid DNA residue, host protein residue, host DNA residue, transfection reagent residue and other impurities in the process study, if applicable. It is necessary to strengthen the in-process control test or quality study in the purification process, such as bioburden, endotoxin, physical titer, infection titer, etc.

　　3.3 Process Validation

　　Process validation shall be executed after the manufacture process is determined to verify each step. If applicable, it may include validation of each step of cell expansion and vector manufacture, validation of storage conditions and time of intermediate products, validation of impurity removal, aseptic process validation, number of cycles of chromatographic column and filtration membrane, validation of sterilizing filters and transportation validation. The process validation proved that the manufacture process and its production can be carried out continuously and stably within the set range of process parameters, the yield and recovery rate of viral vector shall be relatively stable, and the residual nuclease, host cell protein, host cell DNA, plasmid DNA, cell debris, transfection reagents and other impurities shall be effectively removed to the level below the specification range or subject to validation studies.

　　Encourage stakeholders to establish the manufacture process matching the upstream and downstream scale. If combined batch and/or split batch occur during the manufacture process, it is necessary to conduct sufficient validation study in combination with the actual manufacture conditions and develop the principles of combined batch and/or split batch and the specific operating specifications based on the study results. The samples used for batch combination shall be identified as qualified samples through inspection before batch combination.

　　4. Quality Study and Specification

　　4.1 Quality Study

　　Encourage to use advanced analytical methods to carry out product quality study from multiple aspects. Analytical methods shall be evaluated and validated to ensure the accuracy and reliability of the results.

　　It is generally recommended to use multiple representative batches for quality study. Common test items include appearance, viral vector morphology, identification, integration characteristics (if applicable), viral vector titer, biological activity, purity and impurities (such as replication competent virus, residual elements, adventitious agents). Based on the study results, critical quality attributes could be identified.

　　4.1.1 Identity and Sequence Confirmation

　　The viral vectors could be characterized from the viral particle level, protein level and nucleic acid level. In terms of the viral particle, electron microscopy, immune serology and other methods can be used for analysis and identification. At protein level, analytical methods such as protein electrophoresis and immunoblotting can be used to analyze the capsid protein, target gene expression, protein expression profile, immuno labeling and phenotypic characteristics of the vector. And regarding to nucleic acid, whole genome sequencing of viral vectors is recommended to confirm the sequence. Target gene and regulatory element sequence shall be fully analyzed to confirm the sequence consistency with theoretical sequence. For integrated viral vectors, the genomes of cells integrated with vector sequences can also be sequenced after the viral vectors transduction to verify the accuracy of vector backbone and target gene sequences. Restriction enzyme mapping, polymerase chain reaction (PCR) and other methods can also be used to identify the viral vector and target gene sequences. Appropriate positive and negative controls shall be set for identification test.

　　4.1.2 Integration Features

　　For integrated viral vectors, it is recommended to use an appropriate method to study the typical characteristics of vector integration into the target cell genome, including dominant insertion site, insertion copy number, and abnormal growth of dominant clones. Focus on whether there is preferential integration site into the target cell genomic oncogenes and other potential carcinogenic risks.

　　4.1.3 Viral Vector Titer

　　Titer is an important test item for biological activity and quantitation of viral vectors, which can be used for process monitoring and product release. The selection of analytical method shall meet the requirements such as sensitivity, accuracy, and precision. It is encouraged to use multiple methods for titer detection and evaluate the correlation of detection values using different methods. The titer detection includes physical titer (total particle number of viral vector) and transduction titer (infectious particle number of viral vector). In the study, it is necessary to use commercial reference standard or in-house generated reference control to calibrate the test results. The ratio of infectious particles versus total particles in the viral product can be used to evaluate the quality characteristics between different batches and between different stages of vector manufacturing.

　　Physical titer: Quantification of specific proteins/nucleic acids from viral vector can be used to estimate the number of viral vector particles (physical titer). For example, for HIV-1 derived lentiviral vectors, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect p24 protein so that to determine the physical titer. The detection results can be expressed as p24 protein content/ml or particle number/ml. The interference of free p24 to the analytical method shall be evaluated. In addition, the quantification of vector genome copy number can also be used for the estimation of physical titer, and it is recommended to focus on the interference of exogenous DNA during detection. When applying new technology, such as the virus counting method, Nanoparticle Tracking Analysis method, field-flow fractionation-multi-angle light scattering method, etc., the applicability of the method for different type of virus shall be assessed.

　　Transduction titer: Cell-based ex vivo assay can be used to detect the infectivity of viral vectors. Generally, after virus infection and cell culture, the transduction titer of virus vector could be calculated by quantitative PCR of cell genome, flow cytometry, tissue damage/cytopathic effect or plaque formation. The detection results are usually expressed in transduction units (TU/ml), half tissue culture infectious dose (TCID50) or plaque forming unit (PFU).

　　4.1.4 Biological Activity

　　Generally, it refers to the ability of target gene transfer into the target cell and sequential biological function after target gene expression, of which the gene transfer ability is also related to the transduction titer of viral vectors. Biological activity study is carried out throughout the lifecycle of process development. It is recommended to establish the biological activity detection method at the early stage of CMC development; at the stage of BLA and marketing, it is recommended to determine the biological activity detection method related to the realization of intended function (replacement, compensation, blocking, correction of specific gene action, etc.) as far as possible according to the mechanism of action and quality attributes of the viral vector and establish appropriate reference standard when necessary. Since the biological activity of viral vectors may be reflected in the final products of cells, the biological activity of viral vectors can also be evaluated in combination with the biological activity of the final products.

　　4.1.5 Purity and Impurities

　　(1) Viral vector-related impurities: Typical impurities related to viral vectors may include mis-packaged particles, defective interfering particles, non-infectious particles, empty capsid particles, aggregates, and replication competent viruses, etc. It is recommended to use appropriate methods, such as high performance liquid chromatography, electrophoresis, capillary electrophoresis, UV absorption spectroscopy to detect these impurities. The purity and impurity of the viral vector can also be reflected by the ratio of infectious particles to total particles in the viral vector. In addition, at the nucleic acid level, the deletion, rearrangement, hybridization or mutation sequence and other nucleic acid related impurities shall also be considered. The quantity of impurity could be reported as relative quantity (percentage of total content), which shall be included in specification when necessary.

　　(2) Detection of replication competent virus: when replication-deficient or conditionally replication competent viral vectors are used, the replication-competent virus that may be produced during the manufacture process shall be detected at an appropriate stage of the process, and the standard limit for the residual amount shall be determined according to the dose of the final cell product and the risk of viral vector etc. For replication competent viruses that related to product safety, it is necessary to study and establish the sensitive detection methods by referring to relevant research experience or literatures. Common detection methods include indicator cell culture method and direct qPCR method. Samples to be tested include viral vector harvest bulk, end-of-production cells and final cell products harvested during the manufacture of viral vectors. Focus on the method operating, sample size, negative control, positive control, detection markers, detection limit and criteria setting for each detection method. It is recommended to design specific detection markers according to the virus packaging system used. If applicable, it is encouraged to simultaneously employ more than two assays based on different principles or for different biomarkers in the study, to improve the detection capability of replication competent viruses. When the indicator cell culture method is used, it is necessary to select reasonable expanded cells and indicator cells. The inhibitory effect of the test sample on the indicator cell growth shall be considered. The reasonable titer of test samples shall be determined. It is recommended to set a control group for interference evaluation.

　　Replication competent retrovirus (RCR) detection: According to the current analytical technology, it is recommended to use sensitive indicator cell to culture γ-retroviral vector for RCR detection. Test sample could be cultured with RCR amplifying cells so as to amplify the RCR to the greatest extent. After several cell passages, take an appropriate amount of supernatant and inoculate it to RCR indicator cell, followed by cytopathic colony calculation or RCR marker detection for RCR determination.

　　Detection of replication competent lentivirus (RCL): According to the current research technology, it is recommended to use indicator cell culture method for RCL detection of lentiviral vectors. For HIV-derived lentiviral vectors, the HIV viruses which meet the test requirements, such as those lacking auxiliary genes and having replication capacity, could be considered as positive controls for RCL testing. The structure and manufacture process of positive control viruses shall be studied and evaluated, and proper operation and use shall be carried out in a controlled environment. In terms of RCL detection indicators, it is generally believed that the detection of p24 protein, reverse transcriptase activity, psi-gag and VSV-G sequence may reflect the presence of RCL, and appropriate detection indicators shall be selected considering the characteristic and study of viral vectors.

　　(3) Process-related impurities: may include residual host cell proteins, non-target nucleic acid sequences, auxiliary viral contaminants (such as infectious viruses, viral DNA, viral proteins, etc.) and reagent residues used in the process, such as cytokines, growth factors, antibodies, transfection reagents, magnetic beads, nucleases, serum and solvents.

　　Non-target DNA residues, which may include residual host DNA and plasmid DNA co-purified with viral vectors, are common process-related impurities. Non-target DNA may also be co-packaged inside the capsid of the viral vector during the packaging process, which may adversely affect product quality and safety. It is recommended that the process be optimized to reduce this contamination. If necessary, the residual non-target DNA sequence could be confirmed and quantified. When the production/packaging cells are tumor-derived cells, tumorigenic cell lines or cells carrying oncogene sequences require high attention. On the basis of process optimization to reduce residue quantity, it is also recommended to control the size of non-target DNA fragments (recommended to be less than 200 bp) and conduct special monitoring on the known high-risk genes. For example, when HEK293T cells are used for lentiviral vector manufacture, it is necessary to develop a method with sufficient sensitivity and specificity to detect the residues of adenovirus E1 and SV40LT antigen sequences.

　　4.1.6 Others

　　Microbial contaminants: detect the possible introduced contaminants, including adventitious virus, bacteria, fungi, mycoplasma, and bacterial endotoxin.

　　Physicochemical test: The conventional physicochemical test items may include appearance (color, clarity), visible particles, sub-visible particles, pH, quantity, osmolality, etc.

　　4.2 Specifications

　　Specifications are an important part of quality control, including the test items, the methodology of testing and the acceptance criteria for each test items. The testing stages generally include release testing and/or process control.

　　According to the current knowledge, the test items of viral vector specification usually include appearance, identification, purity, quantity/titer, biological activity, impurities, sterility, bacterial endotoxin, mycoplasma and adventitious viral agents. The test methods shall be studied and validated to ensure that the test results are reliable and accurate. If applicable, try to establish controls/standard materials to carry out corresponding quality study for quantity/activity calibration, and to determine storage conditions. In general, the justification of acceptance criteria includes product design, quality study, process development, validation study, methodological study and validation, multi-batch testing and stability results, and reasonable statistical methods.

　　(II) Non-viral Vector Gene Modification Systems

　　Non-viral vector gene modification systems can be delivered into cells by physical, chemical or biological transduction. After entering target cells, they act by transcription, cleavage, translation or other ways. The active components are DNA, RNA or proteins, and may also be a combination of nucleic acid and protein components.

　　1. Molecular Design

　　Design of non-viral vector gene modification systems determines the specificity, accuracy and efficiency of target gene modification or target gene expression, and also affects the safety and efficacy of final gene-modified cell products. It is necessary to analyze and balance the advantages and disadvantages of design and modifications during construction.

　　For DNA gene modification systems, attentions shall be paid to gene transduction efficiency, off-target efficiency, insertion mutations, integration sites and copy numbers of target genes in target cells during the development process. Design strategies include gene codon optimization, modification of chromosomal homologous sequences, modification of GC-rich region sequences, and use of signal peptides and rational promoters. At present, circular DNA gene modification systems commonly used for ex vivo gene modification include plasmids, minicircle DNA, and nanoplasmids. Selection of different types of circular DNA may focus on convenience of preparing highly purified vectors, vector recombination, and effects of epigenetic modification of bacteria-derived DNA sequences in target cells on target gene expression. In addition, special DNA fragments can be introduced into circular DNA gene modification systems to form free vectors, such as vectors containing EBV-derived cis-acting DNA fragment OriP and trans-acting EBNA1 gene. For such vectors, attentions shall be paid to effects of species and cell types on the function of free vectors, and effects of vector recombination and cis-acting/trans-acting DNA fragments on target gene expression.

　　For RNA gene modification systems, mRNA is an example. According to current research data, mRNA design may have a significant impact on its stability and biological activity in target cells. During construction, attentions can be paid to the type and proportion of base modifications, 5’-cap or cap analog structure, untranslated region sequence, Poly(A) tail structure and self-amplifying elements (if any). Meanwhile, codon optimization, regulation of inter-base force and modification of advanced structure can be performed to achieve expected functions.

　　For gene editing systems, it is recommended to optimize sequences of guide sequences, target genes (if any) and gene editing enzymes and their ratios. Through gene editing studies in certain cells, specificity of gene editing enzymes and target sequences can be confirmed, and the best target-binding sequence (e.g., sgRNA sequence) is obtained. Actions shall be taken to reduce off-targeting, insertion mutations and adverse effects on target cell genomic stability. For transposon systems, it is recommended to consider the specificity and distribution trend of integration sites, as well as genomic mobilization. Sequences of the transposon, transposase and corresponding regulatory elements shall be optimized, and the ratio of transposon/transposase shall be properly set.

　　2. Materials for Production

　　The basic principles can refer to the relevant requirements for viral vector gene modification systems.

　　For the raw materials prepared with recombinant technology or biological/chemical synthesis technology, it is necessary to specify the process and quality control situation, especially the analysis of safety-related impurities that may be introduced during the production process. For enzyme reagents used in the manufacture process, it is recommended to focus on the functional activity of enzymes, such as the fidelity and activity of DNA polymerase and RNA polymerase used, enzymatic hydrolysis of digestive enzymes, non-specific digestion conditions of enzymes, and the purity of enzymes, impurities introduced in the production process, etc. For nucleotides, 5 '-caps or cap analogues and other raw materials, the overall quality shall meet the manufacture requirements. It is recommended to concern the identification, concentration, purity and impurities. In the manufacture process, it is recommended to avoid using cesium chloride, ethidium bromide, chloroform and other toxic substances, and avoid using animal derived tryptone, nuclease and other raw materials that may introduce adventitious factors and other risks.

　　For materials used as delivery systems, the critical raw materials involved in their manufacture (lipids, cationic polymers, etc.) require adequate screening and quality control.

　　3. Manufacture Process

　　The basic requirements are same as those for viral vector gene modification systems. For the direct manufacture of cell products after ex vivo gene modification, multiple-time and large-scale manufacture is required, and the specific situation is described as follows.

　　3.1 DNA-like Gene Modification System

　　In the case of plasmid DNA, the manufacture steps generally include microbial culture and fermentation, bacterial collection, bacterial lysis, plasmid purification, concentration and filling. During process study and determination, it is necessary to explore and optimize the key process parameters and establish a stable manufacture process. Key process parameters may include fermentation medium composition, fermentation culture temperature, medium composition, feed time and feed amount, dissolved oxygen content, alkaline lysis buffer and neutralization buffer composition, alkaline lysis time, chromatographic column load, chromatographic flow rate, etc. Attention shall be paid to the possible impacts on plasmid structure and function in the whole manufacture process, such as the irreversible denaturation of plasmid possibly caused during alkaline lysis, etc. During the development of the purification technique, appropriate column chromatography fillers may be selected according to the actual plasmid size and nature to remove impurities like host RNA, host DNA, DNA fragments and bacterial endotoxin to the greatest extent. According to the study, set reasonable process controls and acceptance criteria, such as the concentration of plasmid intermediates, supercoiling ratio and impurity residue.

　　3.2 RNA-like Gene Modification System

　　Based on the current research status, the manufacture of RNA generally includes two process routes: ex vivo chemical synthesis and DNA transcription. For the ex vivo chemical synthesis process, please refer to the relevant technical guidelines for chemical drugs. The DNA transcription process route uses the mRNA manufacture process again, which generally uses DNA transcription template for mRNA ex vivo transcription, mRNA capping, dephosphorylation, DNase treatment, mRNA purification and other steps. It is recommended to study and optimize the process parameters and develop a robust process to ensure the correctness of the mRNA sequence, structural integrity, biological activity, and consistency of quality between different batches. The potential impurities introduced in the process shall be studied to clarify the source, removal steps and removal capacity of impurities. In the process study, it is necessary to confirm the key process parameters and control range, such as NTP concentration, transcription time, reaction temperature, feeding ratio of capping reaction materials, chromatographic medium and dynamic loading capacity, and focus on the product recovery rate, impurity removal rate, capping rate, poly (A) length and distribution (if applicable), the integrity of mRNA fragments and the accuracy of the sequence. Reasonable process controls shall be set during the manufacture process, such as mRNA concentration, double-stranded mRNA content, incomplete mRNA content, residual DNA, sterility and bacterial endotoxin.

　　3.3 Other

　　If the gene modification system contains recombinant protein components, relevant technical requirements for production of recombinant protein biological products may be referred to according to specific circumstances.

　　4. Quality Study and Specification

　　4.1 Quality Study

　　Quality study usually includes identification, structural characteristics, physical and chemical properties, biological activity, purity and impurity analysis. It is recommended that studies shall be performed on multiple representative batches. In case of product containing chemically synthesized components and/or recombinant protein components, quality study may be carried out in reference to corresponding guidelines.

　　Identification and sequence confirmation: In the identification study, restriction enzymes can be used for digestion, followed by electrophoresis analysis to observe whether there is a characteristic band pattern; PCR can also be used to analyze whether the fragment size is consistent with the theoretical size. For RNA product, the test sample could be reverse transcribed into DNA for identification, or other suitable analytical method could also be considered. In sequence confirmation studies, it is recommended to carry out fully sequencing, focusing on the sequence correctness of the target gene and regulatory elements. As for RNA product, if applicable, it is recommended to pay attention to sequence confirmation of poly A length.

　　Structure: It is recommended that structural integrity and size uniformity be investigated by suitable methods. For example, for DNA, attentions may be paid to whether there are various structural forms such as single strand, double strand, linear/open loop, loop and superhelix, as well as possible higher order structure. For mRNA, attentions may be paid to the integrity of structure in different regions (such as 5 '-cap or cap analogue structure, poly (A) length), base modification structure, dephosphorylation degree and possible high order structure (such as stem-loop structure). If the functional activity is related to higher order structure, it is recommended to analyze and study the higher order structure characteristics. For complex nucleic acid gene modification systems, it is recommended to carry out structural analysis, such as the particle size, particle size distribution and particle aggregation of the complex structure.

　　Physicochemical properties: It is recommended to carry out the study on molecular weight, nucleic acid concentration/quantity, modification site and proportion (if any), physical properties (such as pH, osmolality) and other aspects.

　　Biological activity: According to the mechanism of action, the study of biological activity usually includes the determination of gene modification efficiency, expression level of target gene, function of target gene expressed product or ex vivo simulated physiological function. It is recommended to firstly select the quantitation method. If the target gene or gene editing product expression and function could be analyzed, it shall focus on whether the expressed product is as expected or been inhibited, and whether the structure meets the design (such as polymer). When ex vivo cell transfection is used, it is necessary to focus on whether the selected cells are suitable and scientifically sound.

　　Purity: Agarose gel electrophoresis, high performance liquid chromatography, capillary electrophoresis and other methods can be used to study the purity. For complex nucleic acid gene modification system, it is recommended to pay attention to encapsulation efficiency and free nucleic acid quantity.

　　Impurities: On one hand, impurities may come from the dissolution of the raw materials, manufacture process and storage process in direct contact with containers and materials. Such as DNA template, enzyme reagents, magnetic beads and other raw materials and added ingredients; also as ethanol, isopropanol and other organic solvents, in addition to host protein residues, host DNA residues, host RNA residues etc.. On the other hand, for the impurities related to gene modification system, which may include deletion, rearrangement, hybridization or mutation sequence and other related impurities, qualitative and quantitative relevant studies are recommended. For DNA gene modification systems, related studies may include open loop/linear DNA content, molecules modified by hypermethylation, etc. For mRNA gene modification systems, relevant studies may include RNA fragments generated by degradation/breakage, mRNA with incomplete capping, RNA with excessive modification, RNA mismatch sequences, RNA oxidation products, etc. In combination with the impurity clearance capability during manufacture process, the residual levels of impurities shall be analyzed using appropriate methods to assess the associated safety risks.

　　Microbiological safety: it is recommended to detect the possible contaminants during manufacturing process, including bacteria, fungi, mycoplasma (if applicable) and bacterial endotoxin.

　　Study on other characteristics: For the modification system using gene editing enzyme tools, it is recommended to continuously focus on the residue of gene editing system in target cell product in quality study and use bioinformatics tools to analyze the structural changes of target cell genome, single point and small-scale gene mutations, as well as the insertion site and copy number of exogenous DNA, to monitor the intracellular retention time of gene editing tools, and investigate the off-target effect and corresponding safety effects.

　　4.2 Specification

　　According to the risk analysis, in combination with the process study and validation, clinical trials and commercial batch quality analysis, stability study data, etc., develop reasonable quality specifications, specify the analytical methods and standard limit range corresponding to each test item and establish standards/references. For common process-related impurities, if the process is sufficiently verified to prove that the impurities can be effectively and stably removed, the impurities can be controlled according to the process, and relevant residual testing items can be considered not to be listed as verification items.

　　Quality control items may include physical and chemical properties, purity and impurities, biological activity, and safety. Among them, the specification for DNA may include appearance, pH value, quantity, identification, sequence analysis, purity, supercoiling ratio, biological activity, sterility test, bacterial endotoxin, impurities, etc. The specification of mRNA may include appearance, pH value, quantity, identification, sequence analysis, mRNA integrity, capping efficiency, poly (A) length and distribution, biological activity, sterility test, bacterial endotoxin, double-stranded RNA residue and solvent residue. The quality control items of common complex nucleic acid gene modification systems include appearance, pH, particle size and dispersion coefficient, osmolality, identification, sequence determination, quantity/concentration detection, biological activity, purity, sequence integrity, encapsulation efficiency, content of each component of complex materials (such as identification and quantity of lipids), free nucleic acid, visible particles, impurities, microbial safety, etc.

　　In terms of methodological study and validation, the basic principles are the same as the requirements for viral vector gene modification systems.

VI. Stability Study and Direct Contact Container/Material Study

　　(I) Stability Study

　　If the gene modification system involves storage, corresponding stability study shall be carried out. The studies are conducted using representative batches of samples in the proposed storage phase and typically include storage, transportation (if applicable) and in-use stability studies. Before the study is conducted, it is necessary to make an overall plan for the stability study protocol, focusing on the samples, direct contact containers/materials, testing time points, testing conditions and analytical test items for each stability study.

　　During the study, sensitive characteristics that can reflect the quality change shall be studied, such as content, integrity, purity, microbiological safety and biological activity. The proposed conditions shall be covered in the study, such as temperature, light, repeated freezing and thawing (for freezing and storage), shaking, etc. In-use stability studies, such as reconstitution or thawing, and compatibility with diluents, shall be performed depending on the actual use. In the study, it is necessary to use the direct contact containers/materials with the same materials as those in actual use.

　　For viral vector-based gene modification systems, it is recommended to focus on the critical quality attributes of viral vectors such as titer, purity, impurities, microbial safety, and biological activity in the stability study. For non-viral vector-based gene modification systems, it is recommended to focus on critical quality attributes such as physicochemical properties, structural integrity and impurities. For example, the ratio of DNA supercoiled structure may affect the transfection rate of DNA, and the mRNA capping rate may affect the structural stability and translation efficiency of mRNA, which is recommended to be investigated emphatically in stability studies.

　　(II) Study on Direct Contact Containers/Materials

　　If storage is involved, corresponding compatibility study of packaging materials shall be conducted for direct contact containers. According to the results of compatibility study and stability study, select reasonable packaging containers.

　　In addition, for the containers and disposable materials in contact with the samples in the manufacture process (such as storage bags, filter membranes, chromatography resin, pipelines, etc.), risk assessment and/or corresponding compatibility study shall be carried out.

VII. References

　　[1] U.S.FDA. Chemistry, Manufacturing, and Control (CMC) information for human gene therapy Investigational New Drug applications (INDs). 2020.

　　[2] China Food and Drug Administration. Technical Guideline for Research and Evaluation of Cell Therapy Products (Trial). 2017.

　　[3] EMA. Guideline on development and manufacture of lentiviral vectors. 2005.

　　[4] China Food and Drug Administration. Technical Guideline for Stability Studies of Biological Products (Trial). 2015.

　　[5] EMA. Quality, pre-clinical and clinical aspects of gene therapy medicinal products. 2018.

　　[6] ICH. Q5A Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. 1999.

　　[7] ICH. Q5C Stability testing of biotechnological and biological products. 1995.

　　[8] ICH. Q5D Derivation and characterization of cell substrates used for production of biotechnological/biological products. 1997.

　　[9] U.S. FDA. Testing of retroviral vector-based human gene Therapy products for replication competent retrovirus during product manufacture and patient follow-up. 2020.

　　[10] EMA. Guideline on quality, non-clinical medicinal and clinical aspects of products containing genetically modified cells. 2020.