(Chemistry, Manufacturing, and Control (CMC) Development and Evalulation For in vivo Human Gene Therapy Products)

　　Center for Drug Evaluation, NMPA

　　May 2022

I. INTRODUCTION

　　With the rapid development of biotechnology such as gene delivery vectors and gene editing, gene therapy products, which may provide new treatment options for refractory diseases, are making new progress in clinical application. In general, gene therapy products mediate their effects by introducing exogenous genes (or gene editing tools) into target cells or tissues to replace, compensate, block, modify, increase or knock out specific genes. According to different ways of gene introduction into the human body, gene therapy can be divided into two ways: in vivo gene introduction and ex vivo gene introduction. In vivo gene therapy products directly introduce exogenous genes (or gene editing tools) into the human body by appropriate vectors to exert their therapeutic effects, while ex vivo gene therapy products generally introduce exogenous genes (or gene editing tools) into cells ex vivo to prepare genetically modified cells or cell-derived products, which are finally infused back to exert their therapeutic effects. Since there are some differences between in vivo and ex vivo gene therapy products in terms of product type, gene vector type and design, targeting needs of vectors, management of starting raw materials, control of purity and impurity levels, production mode and quality risks, the development and the technical consideration may be different between the two types of products, it should be necessary to introduce our recommendations regarding chemistry, manufacturing, and control (CMC) separately.

　　We are issuing this guideline, with complying with the requirements in Drug Administration Law of the People's Republic of China, Provisions for Drug Registration and other relevant laws and regulations, to assist sponsors of in vivo gene therapy products in following the drug development principles and regulatory requirements. The guideline describes our current considerations and general technical requirements for CMC study of in vivo gene therapy products. Applicability of relevant technical requirements to specific product will be evaluated on a case-by case basis. In the process of product research and development, other more effective methods or techniques may be selected based on scientific considerations, which must conform to drug development principles and have sufficient scientific reasons.

　　The CMC considerations provided in this guideline mainly applies to in vivo gene therapy products at the stage of biologics license application (BLA). For drugs at the clinical trial stage, sponsors may carry out the CMC studies by referring to the guideline appropriately, depending on the phase purpose and features of clinical study. Due to the rapid development of biotechnology, especially in terms of product design and quality study, the CMC studies of different types of products are not the same. With the development of technology, the deepening of cognition and the accumulation of experience, the relevant technical requirements in the guideline will be gradually revised and improved.

II. SCOPE

　　In the guideline, in vivo gene therapy products refer to the drugs that achieve the purpose of disease treatment by modification of genetic materials of somatic cells, expression of exogenous gene, manipulation of cellular gene expression or regulation of cell biological features after entering human body. The product is usually composed of vector or delivery system containing engineered gene construct, and its active ingredients are usually DNA, RNA, genetically modified virus, etc., and some bacteria or fungi may also be developed as vectors for application in gene therapy. Common mechanism of action of products are such as transcription or translation of exogenous target genes, expression regulation of intracellular target genes, modification of the genetic material of target cells, etc. The mechanism of action of some products may require the use of protein components, such as various gene editing enzymes.

　　Due to the complex and diverse composition of in vivo gene therapy products, corresponding technical requirements for the components (such as recombinant protein components) covered by the technical guidelines issued before will not be repeated in this guideline, which can still be referred to. Some oncolytic virus products may also have the features of gene therapy products in terms of design and mechanism of action, and this guideline can be appropriately referred to. Considering the differences in manufacturing process, the Guideline mainly cover the products prepared based on biological technology and may not be completely applicable to the nucleic acid type products produced through chemical synthesis process, such as antisense oligonucleotide products and their derivatives. For multi-component or composite products, such as ribonucleoprotein complex, CMC study shall be conducted for each component and combination product respectively, and corresponding technical requirements shall be met. For example, products developed based on enzyme-based gene editing technologies (e.g., CRISPR-Cas, TALEN, ZFN, Meganuclease, etc.), whose active components may be DNA, RNA (e.g., sgRNA), and/or proteins. According to the process features, while referring to this Guideline, the CMC study of chemically synthesized nucleic acid components (such as sgRNA) should also refer to the relevant technical requirements of chemically synthesized products. The CMC study of protein components should comprehensively refer to the requirements in the previously published guidelines related to recombinant protein biological products. In addition, some in vivo gene therapy products may need to be used with specific drug delivery devices or as drug-device combination products. It is recommended to refer to the requirements of relevant guidelines for medical devices for the drug delivery devices or the device part in drug-device combination products.

III. General principle

　　The CMC study of in vivo gene therapy products should meet the relevant requirements of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the Chinese Pharmacopoeia), especially the requirements of the “General Chapter of Gene Therapy Products for Human Use”. The manufacture of gene therapy products for human use shall comply with the relevant requirements of Good Manufacturing Practice and its appendices. Due to the special active ingredients and mechanism of action of in vivo gene therapy products, the research and development, production, use and waste treatment of products should also meet the requirements of biosafety related regulations.

　　1. General Requirements for Product Design, Development and Manufacturing

　　For the CMC study of in vivo gene therapy products, it is necessary to consider the particularity of various products and the applicability of study stage. In the development process, it is encouraged to explore the impact of production materials and manufacturing processes on product quality based on the concept of “quality by design”, establish the correlation between product quality attributes and clinical safety and efficacy, continuously carry out process optimization and quality improvement, and establish the quality control system of the whole process and the management concept of the whole life cycle.

　　In vivo gene therapy products have special active ingredients and mechanism of action. For example, changes in the replication features of viral vectors may cause non-specific infection and spread of viruses. The gene-modified activity of some products may irreversibly change the genetic material or biological features of cells. Therefore, it is necessary to strictly demonstrate the rationality of various steps such as product design, production and quality control, establish risk awareness in the process of product research and development, and develop corresponding risk control strategies based on the risks. For the selection and design of vectors, it is necessary to comprehensively consider the type, replication features, genome integration features, targeting features and off-target risks, scale-up, clinical indications, mechanism of action, administration methods and frequency, immune risks of vectors, etc., strictly control the potential risk of exogenous factor contamination during the manufacturing process, and monitor the potential risk of homologous/non-homologous recombination of vectors, and establish a justified quality control strategy based on the quality study and the identification of critical quality attributes. In the manufacturing process, it is necessary to carry out safety protection for production personnel and environmental safety control, especially for viral vector type products. The cleaning and sterilization of production line shall be fully validated, and cross-contamination between different products and residual contamination between the same product batchs shall be strictly controlled. Waste procedures for raw materials, intermediates and products are established based on environmental and biosafety assessments to avoid active substance diffusion in the environment.

　　2. General development rules

　　The development and production of products shall follow the general rules of drug development, and be gradually improved and continuously optimized. Due to the different study purposes of products at different stages, the development and production requirements at different stages vary.

　　In the stage of applying for clinical trial, the CMC study of the product shall be able to support the implementation of clinical trial. In this stage, it is necessary to identify and control the factors related to product quality risks according to the preliminary study and in combination with the use of similar products, so as to minimize the safety risks of product in human use. For example, preparation and verification of bacterial/viral seed lots and/or cell banks for production, as well as necessary stability assessment and/or study; safety assessment and quality control of materials for production; development and confirmation of the process of clinical samples, and preliminary establishment of intermediate control; quality study of representative samples and establishment of early specifications; development and confirmation of quality control methods, including establishment and confirmation of safety test methods and preliminary establishment of active analytical methods; stability study supporting clinical trials; screening and suitability assessment of container sealing system. Non-clinical study is an important reference basis for evaluating the safety of the product in human use. The comparison or bridging analysis of manufacturing process and quality between non-clinical study samples and clinical study samples should be special interested to support the safety evaluation of clinical study samples.

　　During clinical trials, based on process development and understanding of product quality attributes, it is necessary to gradually clarify the manufacturing process, key process parameters, control items and key quality attributes during the manufacturing process, establish a stable manufacturing process and a good quality control system. During development, the product manufacturing process may change as the process is developed and optimized. Due to the innovativeness of such products, the cognition and study of such products are accumulated during clinical trials, especially at the early stage of clinical trials, which may be relatively limited and the impact of changes on product quality, safety and effectiveness cannot be fully understood. The evaluation and implementation of change plan shall be more cautious. The implementation of various change plans shall be based on the comparability study adapted to the development stage, reasonably assess the impact of the changes on product quality features, and if necessary, conduct appropriate non-clinical or clinical trial bridging study.

　　At the stage of marketing application, the commercial manufacturing process finally determined after process development shall be able to continuously and stably produce the product meeting the target quality, and the CMC study data shall be able to support the safety, effectiveness and quality controllability of the product. Meanwhile, the work plan for continuous validation and optimization of post-marketing manufacturing process should be developed to better ensure product quality.

IV. Risk Assessment and Control

　　The features and manufacturing process of in vivo gene therapy products vary, and there are great differences in the risks of different products in terms of materials for production, manufacturing process, quality control, and stability. Research should identify and evaluate various risk factors in the production based on the features of products and processes, referring to the quality risk management concept of ICH Q8 and Q9, scientifically using risk assessment tools and based on a case-by case basis, and develop the risk control strategy adapted to the product development stage according to the risk assessment results. In addition, previous knowledge of the product or safety data from previous use of similar products also provides an important reference for risk assessment.

　　Common risks include (but are not limited to) the following:

　　1. Materials for production

　　(1) Contamination of endogenous/exogenous factors in production cell, cellular features and genetic stability, cellular gene modification, tumorigenicity of cells, pro-tumorigenicity and oncogenicity risk.

　　(2) Toxicity, immunogenicity and risks introduced by exogenous factors of raw materials for production; safety and immunogenicity of excipients, especially new excipients and compound excipients for human use, compatibility and stability of excipients; stability of physical and chemical properties of consumables for production, sealing and compatibility.

　　2. Active ingredient

　　(1) Historical generation and modification of virus seed of viral vector, vector type and modification method, replication features, genome integration ability and integration tendency of vector, stability of viral genome, infection and expression features, risk of homologous and non-homologous recombination, immunogenicity, pathogenicity, etc.

　　(2) The correctness and stability of nucleic acid sequence, regulatory features of regulatory elements, oncogenicity of genes and regulatory elements, targeting specificity and tissue expression features, on target/off-target risk, genomic integration features and integration mutation, oncogenicity risk, etc.

　　(3) Loss of plasmid from bacterial or fungal vectors, changes in resistance, or changes in biological and genetic features.

　　(4) Targeting specificity and editing efficiency of gene editing tools, and the impact of impurities or degradation products associated with editing tools on specificity.

　　3. Manufacturing process

　　(1) Manufacturing process and process control, process changes and process deviations, risk of contamination and mix-up during the process.

　　(2) Mutual influence of product production and production environment, personnel, etc.

　　4. Product quality

　　(1) Whether the characterization of quality attributes or specifications are comprehensive and sufficient, and whether the quality risks for studies without effective methods are controllable.

　　(2) Whether the analytical method can meet the needs of quality control.

　　(3) Quality monitoring during production, such as the control of the risks of gene recombination and mutation during production. Polymerization, aggregation, inactivation, degradation, microbial contamination and confusion during production and storage.

　　5. Container sealing system

　　Compatibility-related risks (such as adsorption of product, leakage of packaging material) and sealing performance-related risks.

　　Based on comprehensive risk identification and evaluation results, develop reasonable quality risk control strategy. The control strategy should be developed to reduce the product risks. For example, perform quality control for the risks of raw materials, conduct sufficient characterization and validation study on the manufacturing process, comprehensively validate the quality control methods, establish the quality control system for the whole process from raw materials, manufacturing process to release testing, develop reasonable specifications based on the quality study, determine the storage, transportation and use conditions of the product based on the stability study, and select appropriate primary packaging materials based on the compatibility and sealing study. The revision of risk control strategy shall be made throughout the whole life cycle of product. With the accumulation of production experience and the continuous revision and improvement of the understanding of product quality attributes.

V. General Considerations in Product Design

　　The product design shall be based on the clinical needs of the patients, fully consider the expected mechanism of action, process performance, safety risks, efficacy and other factors of the product, and also consider the safety and convenience of the product in storage, transportation and clinical administration. Since the factors need to be considered for the design of different types of products may be quite different, this section mainly puts forward the general factors to be considered for the product design for the current main types of in vivo gene therapy products (e.g., viral vector products, nucleic acid products and bacterial vector products) for reference. The specific product design still needs to be comprehensively considered in combination with the product features. Because different types of products may share certain similarities in function or mechanism of action, the product design may have to cross-refer to considerations for other types of products. With the development of gene therapy and the enrichment of cognition, the considerations in product design will also be continuously supplemented and improved.

　　1. Viral vector type products

　　The viral vector type products in this Guideline refer to the gene therapy products that transfer exogenous gene of interest into human body by viral vectors. viral vectors refer to the viral particles modified to mediate exogenous gene transfer and/or expression, such as adeno-associated viral vectors, adenoviral vectors, herpes simplex virus vectors, etc. According to whether the nucleic acid carried by the vector is integrated into the target cell genome, it can be divided into integrated and non-integrated viral vectors; according to the replication features of the vector, it can be divided into non-replication competent, conditionally replication competent and replication competent viral vectors. The selection and design of viral vectors and exogenous genes shall be determined based on the analysis and study of vector features, mechanism of action, population antibody level, clinical indications, route of administration and frequency of administration (i.e., potential need for re-treatment).

　　1.1 Gene of interest and regulatory element

　　The gene of interest refers to the gene or nucleic acid sequence in the product which mainly plays a role in therapy or regulation and can exist in the form of DNA or RNA, such as coding sequence of functional protein, nucleic acid transcription sequence with targeted effect, etc. For the selection and design of the gene of interest, it is necessary to consider the pathogenesis of disease, the mechanism of action of product, the sequence difference among races as well as the immunogenicity, functional activity and safety of gene expression product. For the gene of interest which is modified from natural sequence by codon optimization, gene mutation, recombination, indel and/or rearrangement, sufficient modification basis shall be provided and the rationality of the sequence design shall be confirmed through ex vivo and/or in vivo studies. Nucleic acid sequences that play the role of targeting binding designed by platform calculation or based on sequence rules, such as sgRNA, siRNA, etc., need to be assessed for the rationality of the design, and the targeting specificity and on target/off-target risks of the sequences should be assessed and confirmed during the development process.

　　The selection and design of expression regulatory elements of the gene of interest shall be determined according to the type of target cells, regulation needs of gene expression and regulation features of the elements, evaluate whether the regulation of regulatory elements on the expression level, expression persistence, expression specificity (if applicable) and other aspects of target genes meets the expectations, and pay attention to the unexpected regulation risks of regulatory elements, such as: distal regulation function of elements, gene knockout risk caused by the recombination of multiple identical elements, the effect of the insertion of regulatory elements on the initiation, enhancement, termination and insulation regulation of cellular genomic genes. In the design, it is necessary to evaluate the oncogenicity/pro-tumorigenicity of regulatory elements, avoid the use of elements with potential oncogenicity/pro-tumorigenicity risk. If the elements are used, the corresponding elements shall be modified to remove their oncogenicity/pro-tumorigenicity features. If there are regulatory elements that control gene expression in a transient or tissue-specific manner, the corresponding regulatory features of the elements need to be confirmed.

　　1.2 General principle for viral vector selection and design

　　Viral vectors are generally selected and designed for the purpose of effectively delivering and expressing target genes, reducing the pathogenicity of vectors and reducing the risks of recombination and mutation of vectors. Common vector modification methods include deletion, recombination and/or replacement of viral genes. The following factors shall be generally considered (but not limited to) when selecting and designing viral vectors:

　　(1) To study the basic infection rate and neutralizing antibody level of wild strains or vector viruses in the population, and to evaluate the effect of human immune response on the in vivo distribution, transduction efficiency and therapeutic effect of viral vectors.

　　(2) Delete the genes or components related to safety risks as far as possible, for example, the genes that may cause disease/tumor, otherwise, the impact of residual genes/components on product safety shall be evaluated.

　　(3) Minimize the non-essential elements of viral vector, or split the packaging gene of virus, so as to reduce the probability of recombination and reverse mutation of viral vector (such as replication-defective vector).

　　(4) Minimize the homologous sequences of viral vectors with human viruses or endogenous viruses, and reduce the risk of recombination to produce new infectious viruses or replication competent viruses.

　　(5) Both the infection-specificity/tropism and gene transduction efficiency of viral vectors have an impact on the safety and efficacy of the product. The tissue or cell tropism of viral vectors should be consistent with the indications.

　　(6) To study the stability of the viral vector gene sequence, and assess the potential safety risks of sequence mutations and their impact on efficacy.

　　(7) To study the effect of modification of viral vectors on their immunogenicity, pathogenicity and other biological features, to assess whether there is a change in sensitivity to relevant antiviral therapies, and whether viral vectors may have reproductive toxicity.

　　1.3 Special considerations in viral vector selection and design

　　1.3.1 Integration features

　　Integrated viral vectors may have more long-lasting in vivo gene expression activity than non-integrated viral vectors, but the integration process may have an impact on the genomic integrity or expression features of human cells. Therefore, the selection is based on the product design requirements. For integrated vectors, safety screening and/or design modification should be performed for vectors using currently known technical methods to reduce the insertion risk as far as possible, and on this basis, the integration pattern of the vector in the genome and the distribution trend of integration sites should be analyzed to assess the risk of gene mutation, gene inactivation/activation, or cell carcinogenesis caused by its insertion.

　　For non-integrated viral vectors, the theoretical risk of insertional mutagenesis in the cellular genome is relatively low, but adequate studies are needed to assess and/ or confirm the non-integration features of the vector. For example, for vectors such as AAV (Adeno-associated virus) that are generally considered to have non-integrating features, there are still reports of vector integration into the genome in specific cases, so research validation is needed to control the risk. In addition, due to the non-integration of vector, the target gene is free from the cell genome, and the expression time-effect of target gene is easily affected by the stability of vector gene, which may result in the dilution and loss of target gene due to the division of target cells. Therefore, it is necessary to fully consider the mechanism of action of vectors, integration risk, as well as the expression aging of target genes to select and design viral vectors.

　　1.3.2 Replication feature

　　Non-replication competent viral vectors theoretically have a relatively small risk of causing virus spread or uncontrolled infection in vivo, but it is still necessary to confirm the non-replication features of viral vectors by reasonable and reliable detection methods, and comprehensively select and design viral vectors taking into account the impact of vector administration route and tropism on the efficacy and other factors. The design of non-replication competent viral vectors and the selection of packaging systems for the production of viruses should fully consider the possibility of replication competent viruses generated through homologous or non-homologous recombination during the production and use process, establish control strategies for the potential risks of replicable viruses, for example, test the replicable viruses on the samples or final products at an appropriate stage in the manufacturing process.

　　For replication competent or conditionally replication competent viral vectors, due to their replication features that may cause non-specific infection, immune response, cell lysis and other effects, they should be carefully selected in combination with the features, structural design, safety and expected mechanism of action of the vector. The selection and design of such vectors should focus on (but not limited to) the following:

　　(1) It is necessary to analyze the in vivo replication capacity of viral vectors in combination with the mechanism of action and indications.

　　(2)Vectors should avoid inclusion of any known elements with a risk of human oncogenicity.

　　(3) If the viral vector is modified, it is necessary to evaluate the changes in pathogenicity and infection activity of the modified viral vector and control the relevant safety risk factors.

　　(4) The infection and/or replication specificity of the tissues/cells of the viral vector should be adapted to the clinical treatment mechanism. If necessary, consider in vivo inactivation or removal mechanisms designed to increase the vector.

　　1.3.3 Targeting feature

　　The efficacy and safety of viral vector type products are closely related to the infection specificity/tropism of viral vectors and/or the expression specificity of target genes. Therefore, the selection and design of vectors shall consider the tissue/cell tropism of vectors, in combination with the features of receptor distribution, the cell specificity in the regulation of target gene expression and other factors. Different administration routes also have important effects on the targeted distribution of viral vectors. The development process should be based on the selection and design of vectors, ex vivo biological features studies, as well as non-clinical studies, clinical trials and other aspects to confirm the targeting specificity/tropisms of vectors, the expression activity and expression regulation of target genes (if applicable).

　　2. Nucleic acid type products

　　Nucleic acid type products in the Guidelines refer to the products that the active ingredients of nucleic acid with specific function enter human cells through physical or chemical mediation and undergo transcription, cleavage, translation or play a direct role in the cells. The common active ingredients of the products are RNA or DNA. According to different mechanisms of action, nucleic acid type products can function as single active components such as mRNA, shRNA (Short hairpin RNA), miRNA(Micro RNA), plasmid DNA, and minicircle DNA, and can also function as functional systems formed by the combination of two or more components in RNA, DNA , and proteins, such as CRISPR-Cas systems. Nucleic acid type products often need to improve the transfection efficiency of nucleic acid with the help of certain physical transduction devices (such as electro-transfection) or chemical delivery auxiliary media (such as liposome complex, cationic polymer, cationic polypeptide, etc.). Some peptides, antibodies, ligands, etc. with nucleic acid transport function can also assist in the transport of nucleic acid.

　　The structure of nucleic acid type products, the design of target gene and related regulatory elements shall have a reasonable scientific basis, and the features and action mechanism of various active ingredients of nucleic acid shall be fully considered to make them conform to the intended functions. For the design of nucleic acid type products, the design of target genes and regulatory elements may refer to the general requirements in "1.1 Target Genes and Regulatory Elements" of viral vector type products. In addition, based on the features of the product, the following (including but not limited to) factors may also be considered:

　　(1) For products that function by targeted binding of target gene sequences or gene expression product sequences such as shRNA, miRNA, CRISPR-Cas, ZFN, TALEN, or Meganuclease, the safety risks caused by the targeting specificity and off-targeting of the product should be considered, the homology between target gene sequence and non-target sequence shall be reduced, the relevant nuclease structure shall be optimized, and the specificity of binding or editing shall be improved.

　　(2) For products such as CRISPR-Cas , ZFN , TALEN or Meganuclease that act intracellularly by editing the genome, the risk of gene error repair and genomic structural rearrangement caused by genome gaps or double-strand breaks should be considered. At the same time, the duration of action of editing enzymes and the effect of editing enzyme residues on genome stability and cytotoxicity need to be considered.

　　(3) For transposons and other products with genomic integration features, the integration site-specific or integration site distribution trend of the product, the stability of gene integration, as well as the insertional mutagenesis and tumorigenic risk caused by integration should be considered, and the vector sequence, transposase/transposable DNA ratio, number of transposable genes, transposase expression duration and expression regulatory elements should be reasonably designed.

　　(4) For mRNA protein-like coding sequences, the effects of the type and design of 5’-cap or analogue structure, the sequence and length and length distribution of poly A tail, translational regulatory elements, nucleoside modification type, sequence self-replication ability, and delivery system on the immunogenicity, expression activity, and vector stability of the product should be considered.

　　(5) Whether the type and structure of nucleic acid are adapted to the expression duration in the body, the necessity and safety risks of self-amplifying function of nucleic acid and continuous gene expression, the genome integration function of the product and the necessity of the function of the genome for genetic modification of the target cells.

　　(6) The stability, genotoxicity and immunogenicity of nucleic acid structure and sequence of the product.

　　(7) Nucleic acid sequences avoid containing rumen genes. If possible, non-essential elements and genes for screening (such as antibiotic resistance genes) should be removed as far as possible.

　　(8) Chemical stability, human safety, transfection efficiency of auxiliary media for chemical delivery, and compatibility between delivery media, delivery complexes. If possible, consider enhancing the targeting/tropism of the product in vivo, or reducing immunogenicity through the design and screening of delivery aids.

　　3. Bacterial vector type products

　　Bacterial vector type products in the Guidelines refer to the products obtained by engineering bacterial microorganisms that are genetically modified to act as vectors and are used to express target protein or specific nucleic acid sequence in the human body, such as Salmonella, Listeria and Escherichia coli. The construction of bacterial vectors is generally based on the structure and biological features of wild strains and is completed by transfer into plasmids, episomal vector, or modification of the genome of the strains. Modification of bacterial vectors should be based on a reasonable scientific basis and product mechanism of action, with the goal of reducing or removing the pathogenicity of microorganisms and realizing or enhancing the therapeutic function of vectors. For the design and construction of vector, attention should be paid to the changes in genetic features, biological features, pathogenicity, target gene expression activity, in vivo distribution features of vector, the effect on normal bacteria in human body, environmental safety, horizontal gene transfer and the sensitivity to conventional treatment methods. Modification of bacterial vectors should avoid the use of β-lactam antibiotic resistance genes. For the design of exogenous target gene, refer to the general requirements in "1.1 Target gene and regulatory elements" of viral products.

VI. Materials for Production

　　The materials for production in the Guidelines refer to all the biological and chemical materials used in the manufacturing process, including the starting raw materials (such as cell matrix, bacteria, virus seeds, plasmids, etc.), the materials used or added in the manufacturing process (such as culture medium and its added components, tool enzymes, purification fillers, buffers, etc.), excipients, and the consumables for production (such as culture bags, liquid storage bags, pipette lines, filter membranes, etc.). The materials for production are closely related to the quality, safety and effectiveness of products, and the materials suitable for product features and stable supply shall be selected through risk assessment. Standardize the establishment of quality management system for materials for production, including risk assessment, supplier audit, risk and quality control, to ensure product quality and reduce the quality risks related to materials for production.

　　1. Starting raw material

　　There are differences in the types of in vivo gene therapy products, and there are some differences in the initial raw materials for production. The common initial raw materials include plasmid DNA, bacterial seed lots, virus seed lots, and toxigenic cell strains/banks. The starting raw materials shall have clear source and complete traceability information. Generally, the warehouse building management shall be conducted according to the requirements of Chinese Pharmacopoeia. The quality control of starting raw materials shall be determined based on the risks in terms of type, features, historical traceability information, preparation process, product type, manufacturing process and administration route, and quality control shall be adapted to its risks.

　　1.1 Plasmid DNA

　　Plasmid DNA used for production of in vivo gene therapy products can be transiently transfected for production of viral vectors, transcribed ex vivo for production of RNA nucleic acid products, or transfected/transformed for construction of bacterial seeds, viral seeds and cell lines. The structure, gene and element design of various types of plasmid DNA should conform to the general principles and intended use mentioned above. The source and sequence of the original plasmid used for the construction should be clear. The construction results of each step should be confirmed during the construction process. Plasmid sequences, especially those as active components of the final product, should not contain genes or elements with potential tumorigenic risk, and such genes/elements should be replaced or modified if necessary. In order to avoid the abuse of antibiotics and the risk of allergy caused by antibiotic residues, it is recommended to avoid the selection of β-lactam antibiotic resistance gene as the screening gene of plasmids.

　　Except for the disposable plasmids in the construction of bacterial seed lot, viral seed lot or cell line, the plasmid DNA of starting raw materials shall be prepared based on the bacterial seed lot system by validated fermentation and purification process, and the production scale of plasmids shall be adapted to the scale of subsequent production steps. The specification of plasmid DNA generally includes plasmid identification, content, purity, confirmation of complete plasmid or important gene sequence, supercoiling ratio, sterility, bacterial endotoxin, antibiotic residues (if applicable), general physical and chemical properties (such as pH value), process-related impurities (such as host bacterial DNA residue, host bacterial RNA residue, host bacterial protein residue, etc.), and specific items should be determined according to the preparation process and use analysis of the plasmid. Each batch of plasmid is released for use in product manufacturing. Plasmid storage stability should support manufacturing at subsequent manufacturing steps.

　　Plasmids for single use in the construction of bacterial seeds, viral seeds or cell lines may not be prepared by bacterial seed lot system, but the plasmid sequence shall be confirmed to avoid the risk of contamination.

　　Transient transfection of plasmids for viral vector packaging should take into account the general requirements for viral vector design described previously. In order to reduce the risk of replicable or pseudo wild-type virus generated by recombination during virus production and use, it is recommended to preferentially select the virus packaging system verified to have a higher safety level, appropriately split the essential elements for virus packaging into different plasmids, remove non-essential viral genes as far as possible, and reduce the homology between plasmids, and between plasmids and packaging cells and wild virus sequences. For example, in the construction of AAV packaging system, the structural genes of AAV are separated from the transcription units of different plasmids, and the similarity between Rep/Cap gene sequence and sequences such as ITR is reduced as much as possible.

　　1.2 Bacterial seed lot

　　Bacterial seed batch can be used for the production of nucleic acid type gene therapy products (such as plasmid DNA, minicircle DNA, mRNA, etc.), and may also be used as bacterial vector gene therapy products after culture. The origin of bacterial strains shall be clarified, the preparation process of bacterial seed lot shall be clear and complete, the use of human-derived or animal-derived raw materials shall be avoided as far as possible in the preparation process, and the monoclonality of seed lot shall be confirmed.

　　The preparation and verification of bacterial seed lot shall comply with the requirements in General Chapter "Management and Quality Control of Bacteria and Viruses for Production and Verification of Biological Products" and "General Chapter of Gene Therapy Products for Human Use" of Chinese Pharmacopoeia. The verification of seed lot generally includes colony morphology, identification, staining microscopy, growth features, biochemical reaction features, survival rate, antibiotic resistance, electron microscopy, plasmid sequencing, restriction map, expression and/or activity analysis of transgenes (if applicable), etc. The bacterial seed lot shall be free of other bacteria, fungi, phages and other contamination. For genetically modified bacterial vectors, attention should be paid to the phenotype and genotype of bacteria after modification, sequencing should be performed for confirmation of important regions of the genome or genome (such as the introduced therapeutic gene and regulatory elements, as well as the regions within at least 0.5kb flanking the target gene), the insertion site and gene copy number of modified genes should be analyzed, and it is necessary to detect the expression level and functional activity of the target gene. For bacterial vectors modified by attenuation, the features and stability of attenuation should be identified and changes in their antibiotic susceptibility should be detected. For bacterial seed lots for the production of plasmids or bacterial vector seed lots containing plasmids or episomes, the sequences of plasmids or episomes contained shall be confirmed.

　　Considering the changes in bacterial growth features, genome integration or modification genes, expression and activity of target gene, plasmid sequence, plasmid copy number and plasmid loss ratio during the generation process, it is necessary to carry out passage stability study under the conditions simulating or representing the actual manufacturing process. The genetic and phenotypic features of seed lot should be able to meet the production needs.

　　The storage stability of bacterial seed lot shall meet the production demand.

　　1.3 Production/packaging cell bank

　　Production/packaging cell banks are used for virus packaging and production, which can affect the quality and yield of viral vectors and may have the risk of introducing exogenous factors. The residual cellular proteins, DNA and other impurities in cell culture have certain immunogenicity, and the cleavage products or DNA of some cells may also have certain risks of tumorigenicity. In addition, there are many production/packaging cell types that may be applicable in the development of in vivo gene therapy products, such as passaged cell lines/lines with or without tumorigenic features, insect cell lines, etc., and different cells may have different types of risks. For the above reasons, the cell substrate with clear source, clear culture history and no virus contamination should be preferentially selected for virus packaging and production. If the cell matrix containing endogenous viruses is used, the necessity and safety of its use should be evaluated, for example, the human infection activity, immunogenicity, process residue level and viral gene expression activity of endogenous viruses, it is necessary to add the validated virus removal/inactivation process unit in the process and detect the virus residue and activity at an appropriate stage. For the cells with unknown rumen or tumorigenic risk, especially new cell matrix and new cell strains/lines, it is necessary to evaluate the corresponding risk of production/packaging cells by referring to relevant requirements in Chinese Pharmacopoeia and carry out corresponding study when necessary. Due to the relatively high risk of tumorigenicity or oncogenicity of tumor cell lines, caution is recommended. If cells with tumorigenicity are used, the impurity removal performance should be combined with clinical risks and benefits, administration route and manufacturing process (such as residual viable cells, residual oncogenic gene fragments, etc.), evaluate the necessity, rationality and safety of its use, analyze whether the cells carry genes or other factors with oncogenic risk, and control its residual level and gene fragment size when necessary. The use of cells with oncogenicity is generally not recommended. In addition, the residual levels of intact cells, especially of rumen cells, need to be controlled through manufacturing process controls and/or product release.

　　In order to ensure the stability of product quality, it is necessary to establish a bank for the production/packaging cells. The preparation and verification of cell bank shall meet the relevant requirements of General Chapter "Preparation and Quality Control of Animal Cell Substrates for Production and Verification of Biological Products" of Chinese Pharmacopoeia. Verification of cell bank generally includes: cell identification, cell number, survival rate, genotype and phenotype (if applicable), growth features (if applicable), exogenous factors, etc. For cells with unknown tumorigenic features, it is recommended to conduct tumorigenicity test. The exogenous factor detection generally includes sterility, mycoplasma, spiroplasma (insect cells, or plant exogenous components used in the manufacturing process), exogenous viral agents, etc. The detection of exogenous viral agents can be determined after risk assessment in combination with cell modification features in accordance with the requirements in pharmacopoeias. Generally, it should include non-specific viruses, retroviruses, cell species-specific viruses, and potential exogenous viral agents that may be introduced during the culture process of cell strain or cell bank construction. If raw materials of animal origin such as bovine serum and porcine trypsin are used in cell culture history or during the culture process, the detection of viruses of corresponding animal species such as bovine and porcine should be conducted.

　　Based on the production needs, if the cell matrix is genetically modified, such as constitutively expressed viral packaging proteins or replication co-factors, the necessity of gene modification and the applicability of modification method shall be considered. The risk of introduction of exogenous factors shall not be increased in the modification process, and the risk of recombination in the viral packaging process shall be avoided or reduced as far as possible in the selection of modified genes. For example, non-replication competent or conditionally replication competent viral vectors such as adenoviruses should use cell lines that do not contain or have fewer homologous sequences for viral vector production to reduce the risk of recombination during production/packaging. For stably passaged cell strains/lines established by gene modification, the results of gene modification, such as gene sequence, modification site, copy number and expression level, should also be confirmed in the characterization study of cell bank.

　　In order to confirm that the production/packaging cells within a limited number of generations can produce virus vectors with stable quality, it is necessary to carry out cell passage stability study under simulated or representative actual manufacturing process conditions, and determine the production limit passage of cell bank according to the study results. The cells within the passage limit should not affect the genetic and phenotypic features of the packaging virus and can support the production of the virus. For new cell matrix or new cell strains/lines, it shall pay attention to the changes in tumorigenicity during the passage, and conduct tumorigenicity study if necessary; for genetically modified stable passage cell strains/lines, it shall pay attention to whether the sequence and copy number of virus packaging gene are stable during the passage, and whether the amount of substances produced in virus packaging is uniform and meets the quality requirements.

　　1.4 Virus seed lot

　　The viruses for production may include virus vector seeds and/or viruses for packaging, their sources, culture history and construction process shall be clear and complete, the virus features shall meet the production needs and the safety risks shall be controllable after evaluation. It is recommended to use caution for virus seeds with unclear culture history, risk of contamination with other non-target viruses, or unguaranteed monoclonality of strains. If needed, multiple rounds of plaque purification, limiting dilution purification, or by means of DNA/RNA rescue can be used during the construction to ensure the purity and monoclonality of the strains.

　　The virus seeds that can be stored, such as virus vector seeds and viruses for packaging, should be managed so as to reduce the batch-to-batch variation of products. The quality control of virus seed lots shall meet the requirements in “General Chapter of Gene Therapy Products for Human Use of Chinese Pharmacopoeia”. The test items shall be determined in detail according to the features of seed lots, culture history and library establishment process, generally including identification (genomic and immune serological features), virus titer, transcription/expression of target gene sequence (if applicable), biological activity of expression products of target gene sequence (if applicable), contamination of viral vector with exogenous factors (such as bacteria, fungi, mycoplasma, exogenous virus, etc.), replication competent virus (the vector is non-replication competent or conditionally replication competent), etc. In addition, attention should be paid to the specific exogenous factors that may be introduced during the historical passage and construction of seed lot, such as spiroplasma, rhabdovirus and other species-specific exogenous factors that may be introduced by insect packaging cells. The genome sequence of viral vector shall be consistent with the theoretical sequence. In case of any difference, it is necessary to analyze the source of mutation and the stability of viral genome, as well as the impact of mutation on product quality, safety and efficacy.

　　The passage stability study of virus seed lots should be able to represent or simulate the process conditions of actual production, pay attention to the genetic stability of virus seed lots, expression features of target gene, replication features and changes in product quality, and reasonably propose the production limit passage number of virus seed lots based on the results of passage stability study. Carry out the storage stability study of virus seed lots, which shall be able to meet the production demand.

　　2. Other materials for production

　　Other materials for production refer to the raw materials (such as tooling enzyme, antibiotics, culture medium, detergent, purification reagent, etc.), excipients and consumables used in the production in addition to the starting raw materials.

　　The raw materials used in the manufacturing process should meet the relevant requirements of "Quality Control of Raw Materials and Excipients for the Production of Biological Products" in the General Chapter of Chinese Pharmacopoeia. The quality of raw materials should meet their intended use. It is recommended to preferentially select the products approved by drug regulatory authorities or raw materials of pharmaceutical grade for key raw materials. The selection of raw materials shall be subject to sufficient evaluation, and their sources, components, functions, quality, use stage and dosage shall be specified. Non-essential raw materials shall not be used as far as possible to reduce the risks of raw material residues and introduction of exogenous factors. The risk factors related to the production of raw materials and the control of corresponding risks by raw material manufacturers shall be comprehensively reviewed, and reasonable internal control standards shall be established based on risk assessment. If possible, avoid using raw materials of animal or human origin such as serum and porcine trypsin, and try to replace them with serum substitutes or recombinant products with clear components. If it is considered necessary to use this product after study, necessary risk control measures (such as γ-ray irradiation treatment) shall be taken, and a complete quality control system shall be established for the species source, production region, manufacturing process and specification of raw materials to evaluate their TSE/BSE safety risks. It is strictly forbidden to use the raw materials prepared by the animals from the endemic area of spongiform encephalopathy, and the serum/plasma that has not been tested for safety. Evaluate the safety of reagents for production, and avoid the use of β-lactam antibiotics, streptomycin and other toxic and harmful reagents such as ethidium bromide. If toxic or harmful raw materials are used in the production, it should be proved that the downstream purification process can remove them well, or warn of their use.

　　For the relevant requirements for excipients, please refer to Section "1.2.2 Excipients" under "VII. Manufacturing Process".

　　The consumables and containers used in the manufacturing process, such as disposable reaction bag, pipette, liquid storage bag, filter membrane, etc., should have stable physical and chemical features, and the consumables should have good compatibility with the solution in direct contact and production intermediates. Evaluate or study the compatibility of consumables and containers according to the materials of consumables, use stage, supplier study and other factors.

VII. Manufacturing Process

　　1. Manufacturing process development

　　The manufacturing process generally refers to the whole process from the culture/fermentation and purification of cells or bacterial microorganisms to the filling and storage of final products. Due to different product types, the manufacturing process of different products may vary greatly, such as plasmid and viral vector type products. The manufacturing process may involve induced expression, plasmid transfection, virus infection and other operations. RNA and other nucleic acid type products may also be produced by ex vivo transcription process without cell system.

　　The manufacturing process should be developed based on the understanding of the target product quality profile, combined with the correlation between the manufacturing process and product quality, through process study, gradually improve the process, complete the development process from laboratory to commercial scale production, and finally clarify the process steps and key process parameters. If scaled-down model is used for process study, the representativeness of scaled-down model should be confirmed to support that the study results can fully represent the actual manufacturing process. If feasible, it is recommended to adopt the closed manufacturing process as far as possible to reduce the environmental exposure and storage link in the manufacturing process. If there is temporary storage of intermediate products during the manufacturing process, the temporary storage conditions and temporary storage time limit shall be studied and verified. Based on risk analysis, establish the control strategy for the whole process, and reasonably set the control in the manufacturing process, especially the contamination of exogenous factors and the quality control of key intermediates in the manufacturing process. During the life cycle, the manufacturing process should be continuously optimized with the progress of process technology and the deepening of product understanding, and the corresponding comparability study should be conducted for the process changes to ensure the product quality.

　　1.1 Drug substance process development

　　1.1.1 Fermentation/culture process

　　It begins with the manufacturing process of bacterial seed lot fermentation or production/packaging cell culture, and the fermentation/culture process can directly affect the quality of the product. Based on the understanding of product quality attributes, sufficient studies need to be conducted on fermentation/culture conditions, such as scale and mode of fermentation/culture, culture medium and additions, culture temperature, pH, osmotic pressure, agitation speed, pCO2, dissolved oxygen, culture time, inoculation conditions, transfection/infection conditions, harvest time, etc., to develop conditions and parameters suitable for production. For example, for the development of the packaging process of viral vectors, it shall be considered to improve the packaging efficiency and packaging accuracy of vectors and reduce the formation of product-related impurities such as empty vectors, wrong packaging vectors, inactive vectors and free nucleic acids; for nucleic acid type products such as plasmid DNA and minicircle DNA, the sequence correctness, structural integrity, recombination efficiency (such as minicircle DNA, etc.) and conformation of nucleic acid shall be considered. The fermentation/incubation process avoids the introduction of unwanted process-related impurities and tests for impurities and/or adventitious agents at appropriate steps. For packaging production of viral vectors, it is generally recommended that untreated culture harvest fluids be tested for contamination by adventitious agents. In case the adventitious agent test of virus seed or harvest fluid is interfered due to insufficient neutralization of product virus, control cells may be set up in production for adventitious agent test. For non-replication competent or conditionally replication competent viral vectors, a sensitive method should be used to monitor for replicable viruses at the appropriate stage of the process.

　　1.1.2 Ex vivo transcription process

　　For nucleic acid type products prepared by ex vivo transcription of mRNA, the quality of transcription products can be controlled through the study on raw materials, transcription templates and transcription conditions. For the important raw materials for recruitment, such as nucleotides, modified nucleotides, 5 '-caps or analogues, tool enzymes (such as transcriptase, etc.), appropriate quality control should be performed. Attention should be paid to their purity and impurities. Attention should also be paid to the fidelity of transcriptase. The preparation of transcription template should ensure the sequence accuracy and purity of the template and reduce the residual impurities. Ex vivo transcription conditions should be fully studied to improve the accuracy, homogeneity and integrity of transcribed sequences and reduce the formation of side reaction products, such as incomplete RNA, double-stranded RNA, truncated RNA, and long-stranded RNA.

　　1.1.3 Purification process

　　The purification process should be determined based on product type, upstream process and potential impurities, and should be able to stably remove or reduce process- and product-related impurities while not affecting product integrity and activity. If auxiliary virus or packaging virus is used in the culture stage, or there is other potential risk of virus contamination, necessary virus removal/inactivation process steps, such as detergent inactivation, low pH inactivation or virus retentive filtration and other process units should be added in the purification process according to the difference in physical and chemical properties between target product and non-target virus to control the safety risk of residual non-target virus. Monitoring of contamination with adventitious agents and product quality at critical steps may be considered during purification.

　　1.2 Drug product process development

　　1.2.1 Drug product formulation

　　For the selection of dosage form, it is necessary to consider the stability of product storage and transportation, the convenience and safety of clinical medication and other factors. On the premise of achieving the purpose of clinical treatment, it is necessary to design and select the dosage form which is easy to store, transport and use. The formulation shall be designed to adapt to the dosage form, and shall be able to effectively maintain the functional activity and stability of the product and meet the needs of clinical medication. Due to poor stability, some products need to be stored at low temperature, freezing or lyophilization. The formulation of preparations often requires the addition of cryoprotectants, lyoprotectants, active protectants and other excipients with specific functions. On the premise of ensuring the activity and stability of the product, prescription excipients with simple structure, clear components, controllable quality and low safety risk should be selected as far as possible for the design and screening of prescription. The selection basis of excipients shall be sufficient, the function shall be clear, the dosage shall be reasonable, and there shall be corresponding study data support. Try to avoid selecting excipients with high toxicity and high safety risk.

　　1.2.2 Excipient

　　Excipients refer to the auxiliary materials used in the product formulation, such as stabilizers, buffer system, etc., and their selection, dosage and specification should be determined based on the formulation development of the preparation. The quality of excipients should meet their intended function and meet the relevant requirements of "Quality Control of Raw Materials and Excipients for the Production of Biological Products" in the General Chapter of Chinese Pharmacopoeia. The excipients meeting the pharmaceutical standards are preferred. If excipients from multiple sources (such as animal, plant and synthetic sources) or multiple suppliers are used, especially complex excipients such as liposomes, corresponding product characterization and comparability studies should be conducted according to the risk of source and excipient changes, respectively, to prove that the products produced with excipients from different sources are equivalent.

　　If the prescription contains novel excipients that are used for the first time in the human body or used for the first time in the proposed route of administration, the safety of excipients should be systematically evaluated according to the risk factors related to the production of excipients and the corresponding specifications should be established. In the absence of data support for human safety studies, studies should be conducted with reference to the Guidelines for Non-clinical Safety Evaluation of New Pharmaceutical Excipients.

　　Nucleic acid type products often need to be used in combination with certain chemical delivery materials/media to promote or improve the transfection efficiency of nucleic acid, such as nanoparticles, liposomes, cationic polymers, etc. Since the auxiliary delivery materials/media are different from conventional excipients, they have the auxiliary effects of protection, transfection and/or intracellular release during the in vivo delivery of nucleic acid type products and can be considered as functional excipients. There shall be reasonable basis for the selection of delivery material/medium. Generally, the manufacturing process, quality control, human safety and stability of material/medium shall be considered for the material/medium itself. For the delivery system composed of delivery material/medium and nucleic acid, the nucleic acid protection effect, delivery efficiency, intracellular nucleic acid release function and stability of delivery system as well as the process stability and quality change of delivery system shall also be considered. For multi-component delivery systems, quality control and safety evaluation shall be performed for each component of the system, and the interaction between the components of the system and stability of the system shall be studied to determine whether they meet the intended function.

　　1.2.3 Drug product manufacturing process

　　The manufacturing process of preparations shall be determined in combination with the study of product features, preparation formula and dosage form, and the scale shall match the production scale of drug substance, so as to avoid the operation of mixing batches in the manufacturing process as far as possible. If compounding is confirmed, the compounding process should be fully validated and each compounded batch should be manufactured according to the defined process and meet the proposed criteria. In the development process, it may be necessary to pay attention to the preparation method, process operation time, filling accuracy and control of aseptic conditions of the prescription. For special dosage forms such as lyophilization, the lyophilization curve of the preparation should also be studied.

　　For nucleic acid products such as mRNA, during the compounding/encapsulation process of nucleic acid and delivery materials/media, it may be necessary to pay attention to the process parameters that have an important effect on the quality of delivery system, such as temperature, feed ratio, solution concentration, stirring rate, buffer system, mixing flow rate and mixing order, pay attention to the effects of compounding/encapsulation process and purification steps on the compounding/encapsulation efficiency, particle morphology, particle aggregation/dissolution, nucleic acid leakage, impurity residue, nucleic acid integrity and stability, and reasonably set the in-process control items.

　　1.3 Optimization and changes in-process development

　　The manufacturing process may change with the development of the process, such as the change of production site, the change of equipment, the replacement of raw materials, the optimization of process, the expansion of scale, the adjustment of quality control strategy, etc., and the implementation of the change should be based on sufficient comparative study. Comparability studies may be referred to in general principles of ICH Q5E to establish comparability study protocols or bridging plans based on risk assessment of changes. In the risk assessment, it is necessary to consider the influences of the R & D stage of product, the type of change and the processes involved in change on product quality. In the early clinical trial stage, since the manufacturing process has not been finalized and the number of batches is small, the process change can be based on the study data of limited batches for comparability study, but attention should be paid to the changes in safety, purity, impurities, structure, content and other relevant quality attributes; with the accumulation of batch data and the deepening of the understanding of product process and quality, the implementation of the change should be based on a more comprehensive and strict comparability study. According to different types of changes, pharmaceutical comparison may include multiple aspects such as process and in-process control, release testing, expanded quality study and stability. The batch to be studied for comparison should be determined according to the type of change, the importance and variability of the quality attribute, the method of data analysis and the stage of development. Changes with a higher risk/impact are generally considered to require more comprehensive and statistical analysis of batch data in terms of process controls, characterization studies, release testing, and stability. In cases where product quality attributes are not fully comparable, the quality differences observed in the study should be assessed. When existing knowledge or platform experience does not predict the impact of differences in quality attributes on product safety and/or efficacy, or product quality changes are expected to adversely affect product safety and/or efficacy, further bridging study data in nonclinical and/or humans should be considered.

　　Since knowledge and experience with the use of gene therapy products is currently limited, it is generally not recommended to make major changes to the manufacturing process at or after the stage of confirmatory clinical trials, which may increase the complexity of comparability studies and the uncertainty of the results, or even affect the acceptability of clinical trial data. In the process of product development, it is necessary to keep samples of periodic or representative process samples for later retrospective analysis or comparability study.

　　2. Validation and verification of manufacturing process

　　During the process development, according to the purpose of phase study, it is necessary to carry out the confirmation or validation study of manufacturing process adapted to the stage. At the stage of clinical trial application, the manufacturing process of the samples for clinical study shall be confirmed, and the risks in the manufacturing process shall be well controlled, such as the contamination of exogenous factors, cross contamination and product confusion. After the commercial manufacturing process is determined, a standardized process validation should be performed using a representative commercial manufacturing process before marketing. The content of validation study should be determined according to the manufacturing process and risk analysis of the product, generally including all process steps such as cell culture, transduction/transfection, ex vivo transcription, purification and preparation. The number of batches for validation study is related to the complexity and variability of the process, as well as the adequacy of previous process studies and platform experience. Generally, there should be no less than three batches. If there are other special circumstances, it is recommended to carry out communication and exchange with regulatory authorities in advance. The validation study should focus on the controllability of each step of the process, the quality of intermediate products and the stability of process performance, carry out deviation investigation for the deviations occurred during the validation, and formulate the correction plan based on the investigation results. In addition, validation study may also include (but not limited to) sterility validation of manufacturing process, filter sterilization validation, service life study of packing materials and membrane package, transportation validation, cleaning validation, facility and equipment validation, etc. If there is temporary storage of intermediate products during the manufacturing process, the temporary storage conditions and time limit of intermediate products shall also be studied. The results of validation study should be able to demonstrate the stability and control of the process and the rationality of the setting of in-process control items. After marketing, ongoing validation studies should be conducted during commercial manufacturing.

　　For gene therapy products such as viral vectors, if there are virus removal/inactivation units in the process, the virus removal/inactivation effect of the process may be validated with appropriate reference to ICH Q5A, and the safety assessment may be performed based on the validation study results. The validation study shall be able to prove that, the process can effectively remove or inactivate non-target viruses such as packaging virus and endogenous virus used in the manufacturing process, and meanwhile, the activity, structure and other features of target virus vector will not be unexpectedly affected.

VIII. Quality study and specification

　　1. Quality study

　　Quality study runs through the life cycle of products, and continues to supplement and improve with the deepening of cognition and the development of analytical technology. According to different stages of research and development or study purposes, representative batches (such as non-clinical study batches, pilot process batches, clinical sample batches or commercial process validation batches) produced by corresponding processes and samples (such as starting raw materials, production intermediates, drug substance, drug product, etc.) at appropriate production steps may be selected for quality study. At the stage of marketing application, the quality study should generally include at least representative clinical sample batches and commercial process validation batches. If there are differences in quality features between drug substance and drug product, samples should be taken for analysis. For nucleic acid complexes formed by binding with chemical delivery materials/media, nucleic acid, complex components and complete complexes shall be studied, respectively.

　　Validate the critical quality attributes (CQA) of the product through comprehensive quality study. In general, advanced, mature and sensitive methods are selected for quality study to meet the needs of analysis. Due to the possible limitations of the analytical methods themselves, studies using different methods with complementary principles at the same time may be considered. Specific study items should be determined based on the type, mechanism of action and manufacturing process of the product. Common quality study items include (but are not limited to): identification, structure analysis, biological activity, purity, impurities, content, transfection/infection efficiency, general physical and chemical features.

　　1.1 Identification and structural analysis

　　For viral vector type products, the identification and structural study of vectors can be performed at different levels including genome, structural protein and complete viral particles. At the genome level, the genome, target gene and related regulatory sequences of the virus can be confirmed by sequencing, restriction enzyme digestion map, PCR amplification of specific fragments and other methods. If the base or sequence mutation is observed in the study, the primary cause of the mutation should be analyzed. The impact of the mutation on the product safety and effectiveness should be assessed taking into account the effect of the mutation site on the gene function and the genetic stability of the viral vector. At the level of structural protein and viral particles, the expression of structural protein and assembly of viral particles can be confirmed by the separation and identification of capsid protein, immunoblotting, serotype identification of viral particles, structure under microscope , particle size distribution, refraction and other methods. For some viral vectors with complex structure or limited analytical methods, the identification and structure analysis of the virus can be comprehensively carried out by considering the phenotype and activity of the viral vector, as well as the gene sequence analysis after the viral vector infects the target cell.

　　For nucleic acid type products, it is necessary to confirm the correctness of nucleic acid sequence and structural integrity. If nucleic acid has multiple topo-isomers such as single/double stranded, linear, circular and supercoiled, the components shall be identified and analyzed for proportion. The functional activity of some nucleic acid type products may be related to the secondary or higher structure of nucleic acid, such as local hairpin structure. It is recommended to study such structure. The structure and modification of mRNA, such as nucleotide modification, capping modification and PolyA tail, should be identified and confirmed, respectively.

　　For the delivery complex formed by the combination of nucleic acid and chemical delivery material/medium, it is recommended to study the nucleic acid molecule, the structure of the delivery complex, and the interaction between nucleic acid and the delivery complex. For example, the study on complex may include the isoelectric point, structural morphology, particle size and distribution, surface charge, proportion of complex components, nucleic acid recombination/encapsulation efficiency, particle aggregation, nucleic acid release, stability in specific environment, etc. In addition, the identification and content study on the delivery materials/media used should also be carried out.

　　For bacterial vector type products, it is recommended to study the staining features, morphology, colony morphology, culture features and other phenotypic and biochemical features of strains, confirm the genome and/or load plasmid, episomal sequence of strains by sequencing, PCR, restriction enzyme digestion analysis and other methods, especially the feature sequence and engineered sequence, and detect the plasmid size, copy number, plasmid loss rate, mutation of exogenous target gene.

　　1.2 Biological activity

　　Biological activity is an important indicator of product quality and clinical effectiveness. At the stage of marketing, it is necessary to establish a biological activity analytical method that is the same or related to the in vivo mechanism of action of the product for the functional activity study of the product. If the product has multiple functional mechanisms, the corresponding activity should be studied separately. One or more appropriate activity detection methods should be determined as quality control items according to the correlation between the activity and the product action mechanism. If there is a correlation/undertaking of steps or functions between different functional activities, it shall be reflected in the method as a whole as far as possible, such as the correlation between the transfection/infection activity of the product and the gene replacement, compensation, blocking and correction effects of the product in the cells. For the product containing multiple active ingredients, the method shall be established to study the activity of each ingredient, and the possible interference, synergy and other interactions between the active ingredients shall also be considered. The methodological system should try to simulate the in vivo action conditions of the product, select the same or related cell types as the in vivo process, analyze the transfection/infection efficiency, gene expression/inhibition level, activity of the expression product, and other factors related to the action mechanism of the vector or delivery system.

　　For some products with selective delivery features, the tissue/cell tropism, infection specificity or gene expression selectivity of the product shall be studied. Activity analysis methods should consider the establishment of appropriate active controls.

　　1.3 Purity, impurities and contaminants

　　1.3.1 Product related impurities

　　Product-related impurities are products in an unintended, non-functional form that are produced during manufacturing or storage. Potential product-related impurities of viral vector type products generally include non-intact packaged viruses (such as empty capped virus particles, non-enveloped virus particles, etc.), mispackaged virus particles, hybrid virus particles, inactive virus particles, viral particle aggregates, free virus genomes, etc.; common product-related impurities of nucleic acid type products, such as digestion and recombination related sequences, coding error sequences, incomplete sequences, degradation fragments, structural abnormalities and erroneous modification sequences, as well as related impurities generated in the liposome combination process, etc.; product-related impurities of bacterial vector type products may be non-monoclonal strains, loss/rearrangement of plasmids and modified genes, etc.

　　In order to control product quality, it is recommended to separate and identify various product-related impurities, assess their safety risks and consider the control strategy for impurity residues based on the assessment results. In the impurity analysis, the appropriate method can be selected to separate the components according to the differences in the physical and chemical features between the target product and product-related impurities. If necessary, it may be necessary to combine the detection methods with multiple principles to separate and characterize the components of the product. The test results can be expressed in the form of absolute purity and/or relative purity, such as purity by anion exchange HPLC, purity by UV spectrophotometry, purity by gel electrophoresis, SEC-MALS and ratio of infectious particles of viral vector. A variety of unexpected variants may arise during the production and storage of a product, and variants found in the test should be identified and analyzed, with consideration given to their control as product-related substances or product-related impurities based on differences in functional activity and safety between the variant and the target product, with reference to the concept of ICH Q6B.

　　For non-replication competent or conditionally replication competent viral vectors, the most appropriate sample should be selected to detect replicable or wild-type virus produced during the process using a sensitive analytical method. Reasonable residue standard limits are set according to the type of replicable virus, residual risk, process controllability, clinical administration dose, etc. For adenovirus-related gene therapy products, it is generally recommended to control replication-competent adenovirus (RCA) within 1 RCA/3×1010 VP (Viral particles). Replicable viruses should not be detected in products with a greater safety risk such as lentiviruses and retroviruses.

　　1.3.2 Process-related impurities

　　Process-related impurities are mainly introduced by the manufacturing process, such as host cell proteins, host cell DNA, host cell RNA, packaging plasmids, packaging viruses, reagents for production (such as culture media, DNA templates, tool enzymes, purification reagents and fillers, etc.), as well as equipment and consumables such as leachables from production pipelines, packaging, containers, etc. The impurity clearance performance and residual impurity levels of the manufacturing process should be studied to assess the safety risks of residual impurities, and the residual impurities with potential safety risks should be included in the product specifications for control when necessary.

　　If packaging virus is used during the production, the residual level, infectious activity, replication capacity and/or expression activity of packaging virus should be analyzed, the residual safety should be evaluated, and the corresponding control strategy should be developed based on the evaluation results. If tumor cell lines (such as Hela cells), tumorigenic cell lines or cells carrying tumorigenic genes or virus-derived sequences (such as HEK 293T cells) are used for production, while ensuring that there is no intact viable cell residue, the residual amount of DNA and the size of residual fragments shall be controlled, and standard limits shall be reasonably proposed. If possible, it is recommended to control the residual DNA within 10 ng/dose and the size of residual DNA fragments below 200 bp. For known specific transforming sequences with potential safety risks in the product, such as Adv E1A sequence carried by HEK 293T cells, SV40 large T antigen sequence, HPV (Human Papilloma virus) E6/E7 gene carried by Hela cells, residual control should be performed respectively. For viral vectors such as AAV, which are easy to package non-vector DNA into viral particles, the potential risk of packaging relevant exogenous DNA into viral particles is considered in the selection of packaging cells, helper viruses and packaging plasmids.

　　For novel or complex delivery systems, impurities related to the preparation process of polymer excipients such as liposomes, and impurities arising from polymer degradation should also be included in the consideration of impurities.

　　1.3.3 Contaminants

　　Contaminants generally refer to the microorganisms or relevant components introduced in the manufacturing process, such as bacteria, fungi, mycoplasma, exogenous virus, bacterial endotoxin, etc., and the risk of contamination should be studied and controlled.

　　1.4 Content

　　Viral vector type products may determine the viral content by such tests as total particle number, genome copy number, structural protein content, infectious or infectious particle number. In order to ensure the efficacy of the product while effectively controlling the immunogenicity risk of the product, the specification or dose of viral vector type products is recommended to be expressed by the total number of viral particles or genome copy number of corresponding volumes, and the number of active viral particles is ensured by controlling the infectivity and the ratio of infectious particles. Nucleic acid type products can determine the nucleic acid content by detecting the DNA/RNA concentration, copy number, etc., and it is also necessary to study the content of special excipients such as various components of the delivery system (if applicable). For bacterial vector type products, the content can be expressed as the number of living bacteria or the number of colonies. The assay should be calibrated with standards or controls wherever possible.

　　1.5 Analysis of other features

　　Additional characterization may include, for example, appearance, clarity, content of significant excipients, visible particles, subvisible particles, pH, osmolality, fill volume, etc. For integrated viral and nucleic acid type products, the integration site, integration stability and site distribution trend of the product shall be studied to analyze the mutagenic or tumorigenic risks caused by the insertion. For non-integrated viral vectors, it is recommended to confirm the non-integration features of the vector.

　　1.6 Related considerations of gene editing technology

　　At present, all walks of life have a limited understanding of the risks of enzyme-based gene editing technologies such as CRISPR-Cas, TALEN, ZFN, and Meganuclease, and there may be some differences in the editing or cleavage effects of editing enzymes in different cells, while the current study methods still have some limitations in the analysis of editing or cleavage effects. Therefore, for gene therapy products designed based on editing tools such as CRISPR-Cas, TALEN, ZFN, and Meganuclease, a variety of methods are recommended to comprehensively analyze and assess the risk of editing systems. For example, the specific limitations of editing system itself, the specificity of sequence targeting, the analysis of off-target sites, the fidelity and editing efficiency of editing enzymes, the production and quality control of editing system, the advantage screening of editing technology for cell pro-tumorigenicity/tumorigenicity, the immunogenicity of editing system components, the non-specific insertion of editing tool-related sequences, the genomic rearrangement of multi-target editing, and genomic mutations. During the development process, the quality control strategy of the editing system should be improved in combination with the study progress and methodological improvement.

　　The analysis of off-target risk and off-target sites of enzyme-based gene editing tools such as CRISPR-Cas, TALEN, ZFN, and Meganuclease requires a comprehensive judgment in combination with multifaceted information. Due to different sequence design rules and algorithms, there may be great differences in candidate targeted binding sequences such as sgRNA recommended by different sequence design software or platforms. It is recommended to comprehensively compare the design of multiple platforms and analyze and screen candidate sequences for potential off-target sites. In off-target site analysis, bioinformatics tools, sequence homology alignment, off-target system scoring tools and other methods are used for theoretical screening of potential off-target sites, and ex vivo cell simulation tests can be performed with reference to the treatment plan. Karyotype analysis, genome breakage detection, deep sequencing and other methods are used to analyze the potential off-target sites and genome rearrangements. In the simulation test, it is necessary to simulate the temperature, ion concentration, pH value and other conditions for the action of editing system in vivo as far as possible, and consider the off-target situation under the worst conditions such as the heterogeneous sequence of targeted binding sequence such as sgRNA, the isomer of editing enzyme and the concentration of various components of editing system. For the detected off-target site, risk analysis may be performed based on the site location and gene function, and comprehensive judgment may be made in combination with animal or human test data when necessary.

　　2. Specification

　　As an important part of product quality control, specification is the standard used to control product quality determined based on product quality study, and is generally composed of inspection items, analytical methods and standard limits. Specifications generally include specifications for drug substance (if any), semi-finished product (if any), and drug product. Due to process differences, some products may not have a clear production stage of drug substance. In the specification, the stages of drug substance, semi-finished product and drug product should be clearly defined. If some items in the specification cannot be tested in the drug product or drug substance, or the use of samples at other intermediate stages for testing is more conducive to the control of product quality, it may be considered to control the quality of the product by testing appropriate intermediate products.

　　2.1 Test item

　　The inspection items in the specification are generally determined according to the quality study and have significant influence on the safety and effectiveness of product, generally including identification, general inspection items, physical and chemical properties, purity, impurities, content, biological activity and exogenous factor, etc. The specific inspection items shall be determined based on the product type, manufacturing process, quality study, stability and risk assessment. Special dosage forms such as liposomes and nanoparticles should select appropriate quality control items according to specific features, such as particle size and distribution, refractive index, encapsulation efficiency, and surface charge. For non-replication competent or conditionally replication competent viral vectors, control of replication competent or wild-type virus is required. If special container or drug-device combination device is used for the drug product, it is also necessary to add specific test items according to the functions of device. For other test items, refer to the requirements in "general monograph for gene therapy products for human use" in Chinese Pharmacopoeia. Some test items may not be repeatedly tested in drug substance and drug product, but the effect of drug product process on corresponding quality attributes should be considered. For the test items considered important in the study and not included in the specification, there should be sufficient reason or support from the data of validation study.

　　2.2 Standard limit

　　In the specification, there shall be reasonable basis for the establishment of standard limits (acceptance criteria) for each test item. Generally, it is necessary to consider target product quality profile (QTPP), clinical trial exposure, product quality attribute features, batch release test results and stability study. During the establishment of specification limits, when the data analysis of representative process batches is used to determine the control ability of the process to the product quality, it is also necessary to comprehensively consider the stability change trend of the product and the quality exposure of sample batches in the study subjects in non-clinical and clinical studies. The commercial scale production product quality shall be consistent with the samples used in pivotal clinical studies. The specification limit requirements shall generally not be lower than the worst exposure of subjects in clinical studies.

　　2.3 Analytical method

　　In order to realize the effective control on product quality, advanced and mature analytical methods with optimized applicability shall be selected for detection, and it is encouraged to select multiple analytical methods with complementary principles for quality control. Methodological validation should generally be completed before marketing application. However, based on the needs of product quality control and comparative study at different R & D stages, it is recommended to complete the development and validation of methodology before the implementation of confirmatory clinical trials as far as possible. For some quality control methods related to safety and content detection, necessary methodological validation study should be performed before the implementation of clinical trials. If methodological changes occur during development, a bridging study should be performed on the methods before and after the change. For the product with short shelf life or less sample size, the new rapid and trace detection method may be considered as the alternative method of pharmacopoeia method, but it shall be proved that this method has equivalence or superiority to pharmacopoeia method.

　　2.4 Controls/standards

　　In the absence of national standards and international standards, based on the needs of method analysis, corresponding active standards or physicochemical controls can be prepared by themselves according to the preparation requirements of standards, and corresponding quality studies and release tests can be performed. For different R & D stages, the samples produced by the representative process at the corresponding stage can be used to prepare the standards/controls based on the quality control needs, but the bridging study of the standards at different stages should be carried out. The establishment and preparation of controls/standards should meet the general requirements of "Preparation and Calibration of National standards for Biological Products" in Chinese Pharmacopoeia. They should be calibrated according to the purpose and necessary storage stability study should be carried out. In order to improve the traceability of controls/standard substance, it is generally recommended to carry out grading management for reference substance/standard substance.

IX. Stability study

　　For stability study, please refer to the Technical Guidelines for Stability Study of Biological Products (Trial) and relevant requirements of ICH Q5C. The study protocol shall be set according to the features of the product, clinical medication regimen, etc. The study items generally include long-term stability, accelerated stability, study on influencing factors, transportation stability, use stability, etc. The study conditions shall be determined according to the specific storage, transportation and use conditions as well as the study purpose under corresponding conditions. The items to be investigated in the study shall be all-sided and reasonable, especially those having important indicating significance for the stability change trend, safety and effectiveness of the product. The detection method shall be validated and can sensitively detect the stability change features of the product. Product or intermediate samples manufactured using a representative process should be selected for the study and placed in product/sample actual storage containers or other size containers of the same material as the actual storage containers and representative of the worst-case exposure conditions. The shelf life of product/sample shall be determined according to the results of stability study. The study period shall generally cover the actual storage or service life of product/sample.

　　Due to poor stability, viral vector type products are generally stored at low temperature. During the study, it is necessary to pay close attention to the changes in the storage, transportation and the titer (especially infection titer), aggregates and biological activity, limit the number of freezing and thawing of viral vectors and avoid the exposure to the conditions that are easy to inactivate, degrade or aggregate the viral vectors.

　　The stability of DNA nucleic acids is relatively good, but severe environmental conditions and nuclease exposure may still damage the higher structure and integrity of DNA. RNA nucleic acids have poor stability and are more sensitive to Ribonuclease (RNase), and their production and storage should be in a strict RNase-free environment. For nucleic acid delivery complexes formed by combination with chemical delivery materials/media, attention should be paid to the changes in the physicochemical properties and biological activity of nucleic acid and complexes, such as encapsulation efficiency, content, purity, particle size and distribution, surface potential, particle integrity, as well as particle polymerization, aggregation, leakage of encapsulated drugs. In addition, attention should be paid to the stability of chemical delivery materials/medium components such as liposomes in nucleic acid delivery complexes.

　　Bacterial vector type products are generally stable when stored in appropriate cryopreservation solution and at freezing temperature. Attention should be paid to the survival rate of bacterial vectors, stability of transgenes and changes in biological features under the study conditions.

　　In addition to the above features, other physical and chemical properties (such as pH value, osmotic pressure, concentration, insoluble particles, etc.), key components and microbial contamination that may change during storage, transportation and use should also be appropriately investigated during the study.

X. Packaging and sealing container system

　　The packaging and sealing container system generally includes the drug substance (if any), semi-finished product (if any), drug product and the equipped packaging container for diluent. The selection of containers and sealing system should be justified to ensure adequate stability during storage of the sample or product. To avoid the unexpected impact of the storage container or sealing system on the product quality, compatibility studies and compatibility studies should be conducted on the container and sealing system. The compatibility and sealing of the container and sealing system under special conditions should be considered when the study conditions are set, such as the sealing performance under freezing conditions and the compatibility under accelerated conditions. For active ingredients with biological activity such as viral vectors, attention should be paid to the effect of leachable on their activity during storage and use. For the secondary packaging materials with special functions (such as light-shielding materials), the corresponding functions shall be studied and verified. For the products involving special drug delivery devices, such as electroporation device, nasal spray device and needleless syringe, it is necessary to consider the R & D requirements for relevant medical devices as well as the compatibility between drug delivery equipment and products.

XI. Glossary

　　Starting raw materials: the raw materials used to generate or provide components for the active ingredients of the product, such as virus seed lots for the production of viral vectors, transfection plasmids, production/packaging cell banks, plasmids for the production of non-viral vectors, host bacteria, bacterial seed lots, etc.

　　Hybrid virus: refers to the virus produced in the manufacturing process and mixed with other foreign virus genes.

　　Ratio of infectious particles of viral vector: refers to the ratio of infectious activity of viral vector to the number of vector particles.

　　Control cells: refer to production/packaging cells retained in a certain proportion during virus production, without plasmid transfection or inoculation of target virus, with the same medium components as other cells transfected with plasmids or inoculation of target virus, cultured in parallel at the same culture temperature and culture site for a specified period of time. Adventitious agent contamination of this production cell lot was assessed by the interpretation of the control cell adventitious agent testing using a defined method.

　　Nucleic acid complex: Polymer formed when nucleic acid is mixed with auxiliary materials/media for chemical delivery.

XII. References

　　[1] 国家药典委员会.《中华人民共和国药典》(2020年版). 2020.

　　[2] European Medicines Agency. Guideline on the quality, non- clinical and clinical aspects of gene therapy medicinal products [EB/OL]. 2018.

　　[3] European Medicines Agency. Guideline on quality, non- clinical and clinical aspects of medicinal products containing genetically modified cells [EB/OL]. 2020.

　　[4] U.S. FDA. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) Guidance for Industry [EB/OL]. 2020.

　　[5] U.S. FDA. Recommendations for Microbial Vectors used for Gene Therapy [EB/OL]. 2016.

　　[6] CDE. 人基因治疗研究和制剂质量控制技术指导原则[EB/OL]. 2008.