Center for Drug Evaluation, NMPA

　　February, 2023

I. Introduction

　　In recent years, oncolytic viruses, as a tumor immunotherapy product, have attracted extensive attentions. The mode of action of oncolytic viruses includes mainly by lysing tumor cells and activating the body's autoimmune mechanism. With the development of technology and the advances in research, the selectivity and efficacy of oncolytic virus products on tumor cells have been improved, the impact on normal cells has been further reduced, and the safety of products has been further improved. As a result, the variety of oncolytic virus products are increasing rapidly via differences in gene design, manufacturing process, quality control etc. This Guidance is intended to standardize and guide the research, development and evaluation of oncolytic virus products.

　　Based on the current technological development and scientific knowledge, this guidance provides recommendations and general technical requirements for the CMC aspects of oncolytic virus products. Guidance’s applicability for specific product should be assessed case-by-case.. In the process of product development, other more suitable or effective methods could also be adopted according to the actual situation of product development, however, the roles of drug development should be complied and scientific and reasonable basis should be provided.

　　The purpose of this guidance is to guide the CMC development of the oncolytic virus products in NDA stage. In IND stage, the CMC studies should be conducted based on the specific characteristics of the products and the purpose of corresponding clinical stage, taking the recommendations in this guidance into consideration.

II. Scope

　　In this Guidance, oncolytic virus products include wild type, attenuated or genetically modified replication-competent virus products, which can 1) selectively infect tumor cells and/or selectively replicate to lyse tumor cells; 2) simultaneously express exogenous genes to improve corresponding functions; and 3) stimulate the body to generate immune response for therapeutic purposes.

III. General Principles

　　The development and application of oncolytic virus products should comply with the requirements of current laws and regulations from the regulatory agencies. The manufacturing of oncolytic virus products for human use should comply with the basic principles and relevant requirements of Good Manufacturing Practice (GMP). At the same time, the development, manufacturing, use and disposal of oncolytic virus products should comply with the requirements of relevant laws and regulations on biosafety.

　　1. General Requirements

　　Generally, oncolytic viruses are live, replication-competent viruses. Numerous challenges exist in the gene design, manufacturing process, quality research and control, storage and transportation, and clinical use of oncolytic viruses. Therefore, phase-appropriate studies should be carried out to accommodate corresponding drug development stages. To ensure safety, the risk of contamination by adventitious agents should be strictly controlled at different stages for manufacturing materials, in-process controls and/or finished product. Attention should be paid to the risk of gene sequence and/or amino acid sequence mutation after multiple passages of virus should be paid with attention and fully evaluated. To ensure the safety of the product throughout its entire life cycle, appropriate methods and measure should be in place to monitor the mutation risk. Due to the specialness of active ingredients in oncolytic virus products, the risk of contamination and/or cross-contamination in the process of manufacturing should be assessed and controlled. In addition, the potential risk to personnel, animals, plants, microorganisms and the environment in the process of product manufacturing, storing and clinical administration should be evaluated and controlled.

　　2. Considerations at Different Stages of Development

　　The research and development of oncolytic virus products should comply with the general rules of drug research and development, and a phase-appropriate and gradual improvement strategy should be adopted throughout the research and development period.

　　To ensure the safety of investigational drugs used in clinical phase, drug safety should be the main focus. In general, the quality of clinical investigational drugs should be non-inferior to that of non-clinical investigational drugs. In addition, according to product characteristics, the following aspects should also be paid attention to: 1) For the safety of virus itself, aspects like source of origin, construction/screening, passage history and testing, as well as necessary stability study, etc. should all be comprehensively taken into consideration; 2) adventitious agents are the particularly concerned control parameters for oncolytic virus products. The following aspects should be comprehensively considered to avoid contamination by adventitious agents: virus construction/screening, starting materials (e.g., virus seeds, production/host cells), materials of animal/human origin that may be used in the manufacturing process, manufacturing process control and/or finished product. At the same time, it is also necessary to 1) establish and verify the manufacturing process of the drugs for clinical trials and establish preliminary control parameters and limits of the intermediates; 2) carry out relevant quality study and necessary method verification, establish quality specifications suitable for clinical use to ensure the quality of clinical drugs; 3) conduct the preliminary stability studies to support the clinical trials, and assess the suitability of the packaging and container closure.

　　During the clinical trial stage, process optimization and quality study should be timely developed on the premise of not increasing the safety risk of clinical subjects, so that the early pharmaceutical development data could support the late-stage clinical trials. With the in-depth understanding of product quality attributes and manufacturing process, as well as the accumulation of correlation analysis between clinical trial data and product quality attributes, the process steps, critical process parameters and critical quality attributes should be identified and established progressively, and the control parameters and acceptance criteria for manufacturing process should be established, the quality study and specifications should be improved. In principle, it is recommended to verify the manufacturing process and formulation, manufacturing site, source of critical manufacturing materials and product specification before confirmatory clinical trial. The manufacturing process should be relatively stable, and the batch scale and process control should be comparable to commercial manufacturing. Significant pharmaceutical changes that may affect the safety and efficacy of the product should be avoided as possible during or after the confirmatory clinical phase.

　　In the NDA application stage, based on the in-depth study and data accumulation of product characteristics, manufacturing process and quality attributes, the critical quality attributes and critical process parameters of the product should be determined, comprehensive process validation should be completed, and the commercial manufacturing process and formulation should be determined. The test items, analytical methods and acceptance criteria should be specified in the product specification, the analytical methods should be comprehensively validated, and the justification of the acceptance criteria is justified. The stability study and compatibility study of packaging and container closure system should be conducted using representative samples, the scope and data of the study should be complete and comprehensive.

IV. Risk Assessment and Control

　　There are many types of oncolytic virus products with complex genetic design, different biological characteristics and various mechanism of actions. Various types of cells are used in manufacturing/packaging, the manufacturing processes and quality studies could also be different. Therefore, for different types of oncolytic virus products, corresponding control measures should be developed based on their quality risk characteristics.

　　The oncolytic virus products generally include the following quality-related risks:

　　1. Manufacturing Materials

　　(1) Lack of corresponding risk control caused by insufficient study and understanding of the infectivity, pathogenicity and virulence of viruses.

　　(2) The risk of genetic mutation of the virus caused by genetic modification and/or multiple passages of the virus, as well as the risk of carcinogenesis caused by the insertion of the viral gene into the host cell genome.

　　(3) The risk of excessive residual of tumor cell, host cell DNA and host protein caused by lacking knowledge of the cell substrate and inadequate control.

　　(4) The risks of inadequate control of raw materials added to the manufacturing process, especially the adventitious agents of materials of animal/human origin.

　　(5) The risk of safety and immunogenicity caused by novel excipients.

　　2. Manufacturing Process

　　(1) The risks of negative impact on viruses’ physicochemical properties and biological activities caused by manufacturing operations .

　　(2) The risk of contamination/cross-contamination during manufacturing.

　　3. Quality Control

　　(1) The risks generated from inadequate and incomplete quality studies.

　　(2) The risks introduced by limitation or high variability of analytical methods for the testing of molecular variants, non-fully packaged viruses (such as non-enveloped virus particles, empty virus particles, etc.), mispackaged virus particles, non-viable virus particles, virus particle aggregates and biological activity, etc.

　　Based on the above-mentioned quality-related risks, corresponding risk control strategies should be developed. For example, comprehensive study and risk assessment should be conducted for the manufacturing materials related risks; the manufacturing process should be fully studied and validated; comprehensive quality study and quality control should be conducted to accommodate special characteristics of different viruses; the quality control methods should be comprehensively verified and validated; Comprehensive studies on stability and container closure system should be conducted; environmental and personnel risks should be reduced through various measures such as establishing appropriate procedures for waste disposal, and product handling under suitable biocontainment conditions, etc.

V. Manufacturing materials

　　In this Guidance, manufacturing materials mainly refer to the substances or materials used for oncolytic virus products manufacturing, including starting materials (e.g., virus seeds, production cells/packaging cells), raw materials used or added in the manufacturing process (e.g., culture medium and its additives, purification reagents, etc.), excipients, and manufacturing consumables (e.g., culture bags, liquid storage bags, tubing, filter membranes, etc.). The manufacturing materials are directly related to the safety and efficacy of products. Therefore, it is necessary to establish a good and standard quality management system, and carry out risk assessment and quality controls in compliance with relevant requirements in Chinese Pharmacopoeia, etc.

　　1. Starting Raw Material

　　1.1 Virus Seed

　　1.1.1 General Considerations for Virus Selection

　　Generally, the selection of virus is based on the following aspects: clinical purpose, safety (history of viral studies, epidemiological data, molecular structure, previous clinical experience and in vivo safety data, maturity of viral vector design and study basis, etc.), in vivo mechanism of action, biological characteristics (pathogenicity, host range, tumor tropism) and scalability of manufacturing process. When developing with viruses that currently have not been fully-understood, the safety risks should be comprehensively assessed, and appropriate control strategies are required. Current sources of viruses commonly used in the development of oncolytic virus products include wild type, attenuated, and genetically modified viruses.

　　When wild type virus or attenuated virus is used for product development, the source, screening, attenuation operation and passage history of virus should be clarified. In addition, the following aspects also need to be investigated and the risk should be assessed: virus structure and mechanism of action, host range of the virus and its pathogenicity to human body, as well as the virus passage stability. On the other hand, the virulence evaluation test of attenuated virus should be carried out with the consideration of both the attenuation operation and attenuation targets. .

　　When the product is developed by genetic modification technology, the source of the parental virus, culture (if applicable), passage history (if applicable) and genetic information of parental virus should be clarified. In the process of genetic modification, the selection and design of the tools, the operation process and screening methods should be investigated properly, and the rationality of screening results should be explained. It should be noted that following information should be specified: whether the research result meets the expected purpose of genetic modification, such as gene silencing or expression etc.; whether the genetically modified virus generates the risk of unexpected effects in terms of infectivity, replication characteristics, virulence, and pathogenicity, and whether it meets the quality requirements for the development of oncolytic virus products.

　　1.1.2 General Considerations for the Design of Viral Gene Modifications

　　The purposes of viral genetic modification are to improve product’s safety and efficacy by reducing the cytotoxicity on normal cells, enhancing the specificity to tumor cells, and optimizing the tumor microenvironment. Common genetic modifications include (but not limited to) the following:

　　(1) Mutating the viral coding genes which are critical for replication in normal cells.

　　(2) Inserting tumor-specific promoters to control viral early gene expression.

　　(3) Changing the tissue tropism of the virus or the way of virus entering the cell.

　　(4) Inserting exogenous genes into the viral genome, etc.

　　(5) The viral coat protein is engineered and modified to reduce the immunogenicity and antigenicity of the virus itself.

　　Accordingly, for viral genetic modification, the following aspects should be generally considered (but not limited to):

　　(1) Delete or mutate the pathogenic genes related to safety in the virus, such as neurotoxic genes and genes with potential carcinogenic risk.

　　(2) Attention should be paid to following aspects thus to reduce the risk of reverse mutation or homologous recombination: Reducing the homologous sequences between the vector for manufacturing and the packaging cell; reducing the homologous sequences between the packaging plasmids; reducing the homologous sequences between the vector used for manufacturing and human susceptible viruses or endogenous viruses.

　　(3) Genetic stability especially the stability of modified gene of genetically modified viruses should be evaluated. The potential safety risks or efficacy impacts introduced by sequence mutations should be assessed.

　　(4) The following changes of genetically modified viruses should be investigated: growth characteristics, selectivity and infectivity on tumor cells, intracellular replication capacity, and neurotoxicity (if applicable).

　　1.1.3 Establishment and test of virus seed lots

　　In order to ensure the consistency and stability of product quality, the virus used for manufacturing should be managed through the establishment of seed system. During the establishment of virus seed lots, the reasonable screening techniques should be adopted for the monoclonal viruses. For the raw materials that may be used in the establishment of virus seed lots, such as parental genotype virus, plasmid DNA and packaging cells, etc., appropriate study and quality control should be conducted.

　　After the establishment of virus seed lots, relevant tests should be carried out. The test items should comply with the relevant provisions of Chinese Pharmacopoeia or other general guidance. The test items should be set according to the special characteristics of virus seeds. Generally, the test items include identification (gene sequence, serotype, electron microscopic morphology, etc.), virus titer, adventitious agents (bacteria, fungi, mycoplasma, mycobacteria (if applicable), viruses, etc.), parental virus (or wild-type virus), functional test (such as tumor selectivity, virus replication ability, ability to lyse/kill tumor cells, etc.), antiviral drug sensitivity (if applicable), identification and functional test of target protein (if applicable), physical characteristics of virus (if applicable), etc.

　　The storage containers, cryopreservation reagents and cryopreservation conditions of virus seed lots should be investigated and qualified. The seed bank system (secondary or tertiary seed lots) and quantity of seed lots should meet the requirements for manufacturing.

　　1.1.4 Stability of virus seed lots

　　In order to ensure the genetic stability and the passage stability of viruses’ biological characteristics, a standard passage stability study should be conducted for virus seed lots. The conditions for passage study should be consistent with the actual manufacturing conditions or be representative. Following study items are recommended: whole genome sequencing with special attention paid to the analysis of specific gene sequence, product quality (e.g., virus titer, ability to lyse/kill tumor cells, etc.), identification and functional tests on target protein (if applicable), parental virus (or wild-type virus), etc. The passage limit of virus seed lots should be reasonably proposed based on the results of the passage stability study and abided in the actual manufacturing process. It should be noted that some viruses are prone to gene sequence mutation and/or amino acid mutation after numerus passages. Therefore, it is recommended to strengthen the study on virus molecular variants. In general, whole genome sequencing should be performed for the master seed and the viruses reached passage limit in manufacturing, and the changes in gene sequence and/or amino acid sequence of viruses after multiple passages should be focused on. Sensitive and quantifiable detection methods should be adopted to identify all mutations in the viral genome, special attentions should be paid to mutations in the modified or coding regions. The impact of mutations on product safety and efficacy should be thoroughly evaluated.

　　In addition, in order to ensure the stability of virus seed lots during storage, standard storage stability studies need to be conducted.

　　1.2 Plasmid DNA

　　The plasmid DNA can be used to prepare virus seeds or to produce oncolytic viruses by transient transfection. Studies should be performed to verity the sources, sequence information, main elements and functions of various plasmid DNA.

　　In general, the plasmid DNA for virus seeds preparation is only needed once throughout entire construction process, establishment of bacterial seed lots is generally not necessary. Therefore, it is necessary to confirm the integrity and accuracy of plasmid DNA sequence, meanwhile the risk of contamination by adventitious agents should be avoided throughout the operation. For recombinant virus seeds, monoclonal screening, bank construction and test should be performed. For details, please refer to the content above.

　　Generally, bacterial seed lots needs to be established for the plasmid DNA used for the manufacturing of oncolytic virus products by transient transfection. For specific requirements, please refer to “ Guidance for Pharmaceutical Research and Evaluation of in Vivo Gene Therapy Products (draft)”.

　　1.3 Production Cells/Packaging Cells

　　1.3.1 General Considerations for Selection of Production Cells/Packaging Cells

　　In this Guidance, production cells/packaging cells refer to the cells used for virus culture, and generally include the cell substrate used for virus seed construction and the cell substrate used for virus manufacturing. The two cell substrates could be the same or not. In principle, to ensure their applicability and safety, following requirements should be satisfied: clear and traceable source information, good historical safety records, and the experiences of their actual use. ,

　　The following aspects could be considered when selecting cell substrate (but not limited to):

　　(1) Species and tissue origin of the cells.

　　(2) Cells’ sensitivity to the virus and its ability to stably produce the virus.

　　(3) The characteristics of cells and the feasibility of comprehensive test.

　　(4) Convenience and feasibility of the manufacturing process.

　　(5) The possibility of removing the risk factors through downstream purification process. The achievable safety level as well as the utilization experience etc. of the downstream purification process should also be taken into consideration.

　　At present, there are many types of production cells/packaging cells that may be useable in the development of oncolytic virus products, such as cell lines/strains established by continuous passage of normal cell population (e.g., Vero), tumor cell lines/strains (e.g., Hela, A549, etc.), and passaged cell lines/strains carrying tumorigenic genes (e.g., HEK293). In principle, the cell substrate containing endogenous viruses should be avoided. If it is necessary to use them for virus manufacturing, adequate data from safety assessment studies should be provided. If applicable, virus removal/inactivation operation should be used in the manufacturing process to remove/inactivate the endogenous viruses according to the differences in physicochemical characteristics between endogenous viruses and oncolytic viruses, and sensitive methods for detecting such endogenous viruses should be established in the culture or harvest stages. Due to their high risk of tumorigenicity or oncogenicity, tumor cells or cell lines carrying tumorigenic genes should be used with discretion. If tumorigenic cells are used, it is needed to evaluate the necessity, rationality and safety of the use according to clinical risks and benefits, administration route and manufacturing process’s capability on removing impurities (such as live cell residue, oncogenic gene fragment residue, etc.). The oncogenic cells are generally not recommended. For new cell substrate and new cell strain/line, it is recommended to evaluate the relevant safety risks (e.g., tumorigenicity or oncogenicity) by referring the relevant requirements of Chinese Pharmacopoeia, and appropriate studies should be performed

　　If the cell substrate is genetically modified (e.g., to add viral proteins, allow viral replication or packaging, etc.) to suit for the manufacturing needs, the necessity of genetic modification and the applicability of the modification method should be considered. The modification process should not rise any additional safety risk. The selection of the modified genes should avoid or reduce the risk of viral recombination during the viral packaging process as possible.

　　1.3.2 Banking and test of production cells/packaging cells

　　In order to ensure the stability of product quality, the production cells/packaging cells should be banked and managed. The preparation and test of cell bank should comply with the relevant requirements in "Preparation and Quality Control of Animal Cells for Manufacturing and Test of biological products" in Chinese Pharmacopoeia and ICH Q5D. Test items generally include identification, cell number and viability, tumorigenicity and/or oncogenicity (if applicable), adventitious agents, etc.

　　For stably passaged cell lines/strains established by genetic modification, in addition to the above test items, the results of genetic modification, such as gene sequence, modification site, copy number and expression level, should be investigated and verified.

　　1.3.3 Stability of production cells/Packaging Cells

　　During the passage process, standard passage stability studies should be carried out for production cells/packaging cells to ensure that the production cells/packaging cells can stably produce the oncolytic virus with quality that meets the expectation. The conditions for the passage stability study should represent or simulate the commercial manufacturing process, and the study items should generally include identification, adventitious agents, cell growth characteristics, virus manufacturing capacity and virus product quality. In the study on the passage stability of new cell lines, the increasing risk of tumorigenicity and/or oncogenicity with cell passage should be focused on. In the study on the passage stability of cell lines/strains established by genetic modification, attention should be paid to the stability of the genetic modification during the passage, such as the stability of gene sequence, copy number and protein expression. According to the results of the passage stability study, a reasonable passage limit should be established and abided in the actual manufacturing process.

　　In addition, it is necessary to carry out standard storage stability study for cell bank. A reasonable storage stability investigation protocol should be developed. The changes in cell viability, cell growth characteristics and virus manufacturing capacity during long-term storage should be focused on. The storage conditions of the cell bank should be determined based on the study results, and the cell banks under the proposed storage conditions should meet the manufacturing requirements,

　　2. Other manufacturing materials

　　Other manufacturing materials are raw materials used in the manufacturing process other than the starting materials mentioned above, such as culture medium, and additives in cell culture, purification reagents, excipients and critical consumables used in the whole manufacturing process.

　　The quality of these manufacturing materials should comply with their intended use, and their source, composition, use and quality control should be considered during screening. The raw material residues in final product or each appropriate stage of process which would affect the quality of final product should be investigated. Risk analysis should be performed for such residues. When necessary, appropriate limits should be in place for monitoring purposes.

　　In principle, the materials of animal/human origin (e.g., bovine serum, trypsin, etc.) or raw materials that may have unexpected effects on the product (e.g., enzymes, antibodies, cytokines, serum, antibiotics, lysing agents, virus stabilizers, etc.) should be avoided as possible in the manufacturing process. The necessity, rationality and safety of their use should be fully evaluated in the study. The amount and stage of use should be specified. The intended use should be described, and reasonable in-house control specifications should be established according to the certificates of analysis (CoA) of supplier and safety evaluation. If the testing item of some critical raw materials includes microbial limits, it is recommended to sterile filtrate such materials before use to further ensure safety of the product. If possible, it is recommended to use serum substitute with clear components or recombinant products (e.g., recombinant trypsin) instead of bovine serum, animal trypsin, etc., as possible. If necessity of the usage has been confirmed through study, the source (e.g., whether the animals/donor selected are from non-epidemic area), manufacturing process (e.g., whether the materials are irradiated by γ-ray), specification (e.g., whether comprehensive exogenous agent testing is conducted) and TSE/BSE risk of the animal/human origin or raw materials that may have unexpected effects on the product should be fully evaluated, and in-house specifications should be established. In addition, β-lactam antibiotics such as penicillin, as well as streptomycin and other toxic agents such as ethidium bromide, should not be used during manufacturing process.

　　For the relevant requirements for excipients, please refer to 1.2.2 Excipients under section VI. Manufacturing Process.

　　Critical consumables used in the manufacturing process, such as disposable bioreactors, tubing, disposable liquid preparation bags/liquid storage bags, disposable sterilizing filters/membrane filtration kits, etc., should have stable physical or chemical characteristics and good compatibility with direct contacting solution and intermediates. The safety of consumables and containers should be comprehensively evaluated according to materials of consumables, stage of use, supplier CoA, biocompatibility study and other factors.

VI Manufacturing Process

　　1. Process Research and Development

　　Manufacturing process development is based on the current knowledge, research results and risk assessment of the product, as well as the correlation between process parameters and critical quality attributes of the product. The process steps and critical process parameters are gradually established.

　　Process development is often accompanied by process optimization and adjustment. The impact of the changes on product quality should be fully evaluated, and corresponding comparability studies may be conducted. The requirements of relevant guidance such as ICH Q5E could be followed. In the early clinical trial phase, due to the fact that the manufacturing process improvement is still in progress and the number of batches that can be compared with is limited, it is understandable to evaluate the comparability based on the study results of limited batches. It is necessary to pay attention to the impact of changes on product-related critical quality attributes such as safety (residual impurities, etc.), titer, purity, and biological activity. As process development progresses and knowledge of the manufacturing process and quality attributes of the product grows, comparability studies should be more comprehensive. Generally, comparability studies include process performance (process parameters and in-process controls), release testing, extended characterization, and stability studies. Due to the limitation of current knowledge, the variability of some analytical methods may be high, and the correlation between critical quality attributes and clinical efficacy is unclear or difficult to establish. As result, these factors will lead to a greater uncertainty in quality comparability. Therefore, it is recommended to fully evaluate the quality differences observed in the study. When the impact of differences in quality attributes on product safety and/or efficacy cannot be predicted by current knowledge or platform experience, further non-clinical and/or clinical bridging studies should be considered.

　　1.1 Drug substance Manufacturing Process

　　At present, the common manufacturing process of drug substance includes a series of operation unites such as cell culture, virus infection, virus harvest and virus purification, etc. During process development. , appropriate culture methods should be adopted according to the characteristics of virus and cells used for manufacturing. Meanwhile, the controllability of virus quality, the scalability of process, the maturity of technology and the convenience of process operation should also be considered to design and develop the manufacturing process of drug substance.

　　With adequate process research and development, relevant parameters of cell culture and harvest process such as culture time, culture temperature, cell density, multiplicity of infection (MOI) of virus or plasmid transfection ratio (if applicable), and virus harvest conditions should be identified, and appropriate ranges should be established for these identified parameters. In general, in order to ensure batch-to-batch consistency of product quality, in virus inoculation and culture, the same working seed lot should be inoculated and cultured at certain range of MOI according to previous study. The physical and chemical characteristics of virus, virus activity and impurity removal should be fully considered for the development of purification process. The process steps and process parameters should be established based on the study results.

　　1.2 Manufacturing Process of Drug Product

　　1.2.1 Formulation Study

　　The type and quantity of excipients used should be screened in combination with quality study and stability study. During the studies, it is recommended to pay attention to the changes in quality attributes such as viral titer, purity, biological activity, particulate matter and visible particles. For lyophilized preparation, the changes in quality attributes such as appearance, pH value and moisture after lyophilization and reconstitution should also be studied. Oncolytic virus products are usually stored under frozen condition, so the formulation generally contains functional excipients such as cryoprotectant and lyophilization protectant. In formulation screening, comprehensive study on the type and amount of cryoprotectant and lyophilization protectant is recommended. Under the premise of product stability ensured, it is suggested to select the excipients with well-defined components, controllable quality and less safety risk.

　　1.2.2 Excints

　　The quality of excipients used in the drug product should meet their intended functions and comply with the relevant requirements of "Quality Control of Raw Materials and Excipients for the Manufacture of Biological Products" in the General Chapter of Chinese Pharmacopoeia.

　　The selection and amount of excipient should be based on adequate formulation screening studies. . The use of excipients of animal/human origin should be avoided as possible. When usage of excipients of animal/human origin cannot be avoid, adequate studies should be conducted to demonstrate the necessity, safety and rationality of their usage. If a novel excipient is used in the formulation, sufficient studies on manufacturing process, quality and stability should be conducted. In addition, supplier audits should be conducted, corresponding specification/in-house specification should be developed, and relevant studies stated in the Guidance for Non-clinical Safety Evaluation of Excipients for New Drugs should be conducted.

　　1.2.3 Study on Process of the Drug Product

　　The manufacturing process of the drug product generally refers to the process from formulation of the purified drug substance or drug substance formulation (if applicable) to the final formulation of drug product. Formulation, sterile filtration (if applicable), filling, lyophilization (if applicable) and other operating steps should be considered in the process study. Reasonable process steps and parameter ranges should be established according to process development and study. Since oncolytic viruses are live and replication-competent viruses, it is recommended to adopt closed filling as possible to reduce the risk of transmission during the manufacturing process. In addition, it is recommended to match drug product manufacturing scale with that of drug substance, and mixing different batches of drug substance to prepare intermediate or finished product should be avoided as possible. In some cases, the sterile filtration cannot be performed due to the physical properties of virus. Therefore, it is necessary to strengthen the quality control of raw materials for manufacturing and strengthen the process control of the manufacturing process, and establish corresponding measures to control the risk of contamination.

　　1.3 In-Process Controls

　　In order to ensure the reproducibility of the process and the consistency of product quality between batches, and to enhance the risk investigation during the manufacturing, adequate in-process control and acceptance criteria should be established. The in-process controls should be determined based on comprehensive consideration on process characteristics and product characteristics, such as number of cell passages, cell density, cell viability, viral titer, purity and impurity, biological activity, etc. In addition, attention should be paid to the tests and controls related to microbial safety in process control. It is recommended to monitor the safety items such as microbial limit or sterility, bacterial endotoxin, adventitious viral agents, mycoplasma, etc. at the appropriate stage of the manufacturing process. If a rapid detection method is required for some in-process control items, sufficient study and validation should be conducted, and it is recommended to carry out study to demonstrate its comparability with the methods specified in Chinese Pharmacopoeia (if applicable).

　　2. Process Validation

　　The objective of process validation is to demonstrate whether by following proposed process steps and parameter controls, the established manufacturing process can be used to consistently manufacture products that meet the expected specifications. In general, after the commercial manufacturing process has been established, a standard process validation should be conducted using the commercial manufacturing process and with a representative scale prior to the NDA submission. And a complete process validation report should be provided as part of NDA. The number of batches for validation study should be considered according to the process complexity, process variability, adequacy of previous process study and data accumulation, as well as the level of understanding of manufacturing process. Generally, the number of batches should not be less than three batches. If there are other special cases, it is recommended to communicate with regulatory authorities in advance. The results of validation studies should demonstrate the robustness and controllability of the process, as well as the appropriateness of the in-process controls and their acceptance criteria. In addition, validation studies may also include (but are not limited to) validation of media/buffer preparation and storage conditions, validation of service life of chromatographic packing material, validation of sterile filtration, validation of aseptic process, and critical raw materials sterilization performance validation. If the temporary storage/holding of intermediates is involved in the manufacturing process, the validation data supporting the temporary storage conditions and duration should be provided.

VII. Quality Studies and Specifications

　　In the process of product research and development, it is necessary to conduct comprehensive, systematic and in-depth study on product quality. Scientific, justifiable and feasible specification should be established. The specification would be continuously revised as needed and improved to ensure safety and efficacy.

　　1. Quality Study

　　For quality study, samples from representative process batches (e.g., non-clinical study batches, clinical study batches and/or commercial process batches, etc.) and samples from appropriate manufacturing stages (e.g., starting materials, process intermediates, drug substance, drug product, etc.) should be selected for quality study. A series of physicochemical, biological, and immunological analytical methods should be used for study. The quality study should be comprehensive and sufficient, covering all the quality aspects, as far as possibly related to product safety and efficacy.. Quality items generally include identification, structural analysis, biological activity, content, purity, impurities, and contaminants, as well as other characteristics.

　　1.1 Identification and Structural Analysis

　　It is suggested that various techniques and methods should be used to identify the genome, morphology and structures of the virus. At the genome level, sequencing, restriction endonuclease, PCR and other methods can be used to confirm the specific sequence of viral genome, target gene or regulator gene. In general, restriction endonuclease method and PCR can only confirm some characteristic sequences, however cannot obtain the information of other unexpected mutations in the whole genome sequence. Therefore, it is recommended to use several batches of products manufactured from representative manufacturing process for whole genome sequencing. If mutations in gene sequences or locus are found in sequencing, the reasons for the mutations should be analyzed. Also, the impact of molecular variants on product safety and efficacy should be evaluated. Evaluations based on literature research and/or models of their potential impact on protein-protein interactions or functions are also acceptable.

　　The identification of virus morphology and structure is generally analyzed focusing on the particle integrity and protein level, and the viral particle structure, particle size distribution, etc. can usually be analyzed by electron microscopy. Protein identification can be analyzed by protein electrophoresis, immunoblotting, immunoneutralization test (serotype identification), etc.

　　1.2 Biological Activity

　　Based on the mechanism of action, biological characteristics, and purpose of gene modification of the product, the biological activity indicators that can reflect the in vivo mechanism of action should be established. If the product is designed with multiple functions, it is recommended that appropriate potency assays should be established separately. Based on the correlation between activity and the mechanism of action of the product, one or more appropriate activity potency assays should be established as quality release testing items. The biological can be tested using in vitro methods to detect the toxicity/lysis effect of oncolytic virus products on representative susceptible tumor cells and/or virus’ replication ability in cells, or by using primary cultures of animal/human tumor cells. In some cases, when in vitro testing does not fully reflect the in vivo effects of oncolytic viral products, in vivo testing in animals may be considered. For some oncolytic virus products, biological activity testing also requires quantitative and qualitative analysis utilizing transgene expression levels and biological characteristics. For the analysis of biological activity, it is necessary to establish and use appropriate standards to calibrate the relative potency of the test sample or calculate the IC50 value of the test sample.

　　1.3 Viral Content

　　For oncolytic virus products, the viral content can be determined by physical titer (e.g., total count of viral particles, genome copy number, the content of structural protein, etc.) and infectious titer. Physical titer can be assessed by physical, biophysical, or other methods to determine the physical number of particles, or by measuring a representative structural protein with known molecular weight and copy number in the viral particle. Cell-based in vitro assays such as plaque forming unit (PFU) and 50% tissue/cell culture infectious dose (TCID50/CCID50) can be used for infectious titer test. The host range and tissue tropism of virus vector should be considered for the selection of cells used for infectious titer test. Susceptible and appropriate cell lines/strains should be selected. The cells for testing should be banked，and the cell bank should be fully tested following the relevant requirements of Chinese Pharmacopoeia. In addition, it is recommended to study and monitor the ratio of physical titer to infectious titer, which can be used to investigate and understand batch-to-batch consistency of product quality and the robustness of the process, as well as to monitor impurity levels.

　　1.4 Purity, Impurities and Contaminants

　　Due to the complex structure of virus; the tendency to mutate and change of the biological characteristics during the passage process is high; and the characterization methods for different viruses are limited; it is difficult to determine purity with a single indicator. Therefore, based on the characteristics of viral structure and manufacturing process, appropriate analytical methods and test items should be selected for product purity and impurity study, and reasonable specification limits should be established.

　　1.4.1 Product-Related Impurities/Substances

　　Product-related impurities/substances generally refer to product-related unexpected materials generated during the manufacturing process, including molecular variants, non-complete packaged viruses (e.g., non-enveloped viral particles, empty viral particles, etc.), mispackaged viral particles, inactive viral particles, viral particle aggregates, etc. In general, substances that are not product related but may affect the safety and/or efficacy of the product are classified as process-related impurities. It is recommended that an appropriate methods should be used for testing of residues, and safety assessment should be performed for process-related impurities. If necessary, it should be considered to include control of process-related impurities in specification; Substances demonstrated to have no impact on product safety and/or efficacy by study data are classified as process-related substances. Monitoring of process-related substance at appropriate stages should be in place to ensure batch-to-batch consistency.

　　At present, most oncolytic viruses have been modified by deletion or insertion of partial gene sequence, or have been attenuated to improve their selectivity and reduce their toxicity. During the virus packaging or manufacturing, these viruses may form the parental genotype virus (or wild-type virus) through recombination or reverse mutation, etc. In addition, gene sequence and/or amino acid sequence mutations may occur during multiple passages or manufacturing process of the virus. These unexpected molecular variants may change the replication selectivity and oncolytic properties of the product, and may also affect the killing function of the product to tumor cells, as well as affect the expression level and biological characteristics of the inserted gene, etc. Therefore, it is necessary to investigate and control such unexpected molecular variants. It is encouraged to use appropriate and advanced analytical methods for the study and testing of molecular variants, and to establish reasonable specification utilizing nonclinical and/or clinical data.

　　During virus manufacturing, product-related impurities such as non-complete packaged virus, mispackaged virus and inactive viral particles may be generated due to incomplete or incorrect packaging of virus. Combining particle size, biological characteristics and physicochemical characteristics, it is recommended to select analytical procedures with different principles, such as high-performance liquid chromatography, transmission electron microscopy, flow cytometry, enzyme-linked immunosorbent assay, analytical ultracentrifugation, ultraviolet spectrophotometry, PCR, etc.. Based on the results of residue detection, a reasonable quality control strategy should be developed.

　　Viral particles are prone to aggregation and form aggregates, which may lead to potential safety risks. Advanced and quantifiable methods are encouraged to be used for aggregate testing. The risks should be comprehensively assessed in combination with the impact of aggregates on the in vitro biological activity and in vivo effect of the product, and quality control of aggregates should be performed when necessary.

　　1.4.2 Process-related impurity

　　Process-related impurities are mainly derived from the manufacturing process itself, including impurities sourced from starting materials (e.g., cell debris, host cell DNA, host cell protein, etc.), raw materials used for manufacturing (e.g., culture reagents, purification reagents, etc.) and equipment/consumables. Potential process-related impurities should be identified, evaluated, qualitatively and/or quantitatively analyzed, and safety assessment should be performed taking residual levels of process-related impurities into consideration.

　　As the residual host cell DNA may affect the safety of the product, it is generally required to test and control the residual host cell DNA. If tumor cell lines (e.g., Hela cells) or cells carrying oncogene or virus-modified sequences (e.g., HEK293T) are used in the manufacturing, more specific studies should be carried out. For example, the amount of residual DNA and fragment size of host cells should be controlled. It is recommended to limit the amount of residual DNA to less than 10 ng/dose and the residual DNA fragment size to below 200bp as possible. For known specific transforming sequences with potential safety risks, such as E1A and SV40 Large T antigen sequences in 293T cells, E6 and E7 gene sequences in HeLa cells, the corresponding test and control of the residue should be performed.

　　For the cell culture additives used in the manufacturing, such as bovine serum, nuclease, (recombinant) trypsin, Triton X-100, cytokines, etc., the residue should be tested, and the safety evaluation should be conducted.

　　For impurities from equipment/consumables, such as leachable and extractables, chromatographic packing exfoliations, it is necessary to perform a corresponding safety evaluation taking the type and residual level of impurities into consideration, and pay attention to their influence on product safety and efficacy.

　　The acceptable level and limits for these process-related impurities should be justified based on nonclinical and/or clinical data or research experience, as well as industry and regulatory consensus, etc.

　　1.4.3 Contaminants

　　Contaminants refer to microorganisms (e.g., bacteria, fungi, mycoplasma, exogenous viral agents) or other relevant components (e.g., bacterial endotoxin) introduced in the manufacturing. Measures should be taken to avoid and control the introduction of contaminants during the manufacturing process. It is recommended to adopt a strategy of release testing, supplemented by an in-process monitoring.

　　1.5 Other Characteristics

　　Studies are conducted in combination with product types and different dosage forms, which may include appearance, color, clarity, visible particles, sub-visible particles, pH, osmolality, filling/filling variation, moisture (if applicable), content of excipients, etc.

　　2. Specification

　　The specification is generally determined based on the quality study of the product. In the quality study, it is necessary to determine the critical quality attributes. In general, the critical quality attributes should be included in the specification. Due to different manufacturing processes for different products, it is necessary to establish applicable quality criteria for samples at different stages in combination with process characteristics. The quality criteria generally include the specifications for drug substance (if any), intermediates (if any) and finished product.

　　The specification consists of three aspects: test items, acceptance criteria and analytical methods. Based on comprehensive and targeted quality study, the safety and efficacy of the product, as well as the influence of various links in manufacturing, distribution and administration should be fully considered. The testing items and acceptance criteria for product quality should be determined, and a justifiable and feasible specification that can reflect the changes in product characteristics and quality should be established to effectively control the consistency of product quality and batch-to-batch quality.

　　2.1 Test Items

　　The test items of the specification should be comprehensively determined based on the sufficient quality study and according to the product characteristics, quality attributes and process correlation, stability study and safety evaluation. Generally, identification, purity and impurities, content, biological activity, contaminants and general test items are included in specification.

　　For identification test, it is necessary to select the relatively specific method according to the characteristics of the product.

　　For the testing of purity and impurities, methods of different principles are recommended.

　　The biological activity should reflect or be relevant with the expected mechanism of action and in vivo efficacy of the drug.

　　For general tests, appropriate items should be developed according to the formulation and dosage form of the drug product. In addition to the routine items specified in the Chinese Pharmacopoeia, the content test of functional excipients and the testing of moisture in the lyophilized drug product should also be considered.

　　The test items identified as critical quality attributes by study but not included in the specification should be sufficiently justified, supported from validation study and/or have reasonable quality control measures in place.

　　2.2 Specification limits

　　The determination of specification limit is generally based on safety and efficacy. Generally, it should be comprehensively established taking product characteristics, clinical trial exposure, test data of samples at various stages (especially clinical trial batches), variability of analytical methods, and stability studies into consideration. The specification limit should be adequately justified.

　　2.3 Analytical Method

　　The analytical methods in the specification are the basis of product quality control. In order to ensure that the analytical methods used are suitable for the corresponding testing requirements, methodological study should be conducted. For methodological study, appropriate methods should be selected and sufficient validation should be performed. If applicable, after study, the analytical method specified in Chinese Pharmacopoeia should be prioritized. If an non-compendial method is selected, it should be proved that the alternative method is equivalent or superior to the compendial method. The analytical methods can be gradually improved with the development of product study. Before the confirmatory clinical trial is conducted, it is recommended to complete the method qualification or comprehensive method validation to ensure that the quality of the samples used for confirmatory clinical trial is consistent with that of the commercially manufactured product. In case of optimization or change of analytical methods during clinical trials, the differences in methods should be analyzed and justified, and the methods before and after change should be compared to prove the equivalence of the two methods.

　　2.4 Standards/Reference Standards

　　In order to ensure the reliability and accuracy of the test results, it is generally necessary to establish standards/reference standards. It is recommended to use representative samples to establish standards/reference standards according to the quality control requirements at different stages of R&D, and carry out the quality bridging study of the standards/reference standards at different stages. The standards/reference standards in oncolytic virus products is generally used for identification, physicochemical and biological activity assays. The establishment and preparation of standards/reference standards should follow the relevant requirements of "Preparation and Calibration of National Reference Materials for Biological Products" in Chinese Pharmacopoeia. Quality study, calibration and stability study should be conducted comprehensively.

VIII. Testing and Control of Viral Exogenous Agent

　　There may be a risk of viral contamination in the manufacturing of oncolytic virus products. Due to the replication-competent nature of oncolytic viruses, the test and control of adventitious viral agents is challenging. Common control strategies include:

　　1. Control of Materials Used in Manufacturing

　　Cell banks, virus seed lots and raw materials used in the manufacturing should be fully tested and controlled for adventitious viral agents.

　　The test of adventitious viral agents of cell banks can be determined by referring to relevant requirements of Chinese Pharmacopoeia and ICH Q5D and taking risk assessment on cell source, passage or modification history, and raw materials used in the process of bank establishment into consideration. Generally, adventitious viral agents control includes non-specific viruses, retroviruses, cell species-specific viruses, bovine viruses (such as using bovine serum), porcine viruses (such as using animal-derived trypsin) and other potential exogenous viruses.

　　For the test of adventitious viral agents of virus seed lots, neutralizing antibodies can be added to eliminate the influence of oncolytic viruses on the test results. The neutralizing antibodies should be selected to avoid the presence of antibodies that neutralize potential adventitious viral agents in antisera. The amount of neutralizing antibodies used should be the minimum amount that completely neutralizes oncolytic viruses. The concentration of test samples should be reasonable and based on research to avoid the risk of reduced detection sensitivity caused by excessive dilution of test samples. If the test results are interfered due to the inability of neutralizing antibodies to sufficiently neutralize oncolytic viruses, control cells can be set in the manufacturing of virus seeds, and the test of adventitious viral agents of control cells should comply with the requirements of current Chinese Pharmacopoeia. In addition, sensitive detection techniques such as polymerase chain reaction (PCR) and deep gene sequencing technology can also be considered to exclude the risk of specific contamination from adventitious viral agents.

　　The materials of animal/human origin used in the manufacturing should be subject to specific viral test in combination with the source of raw materials, such as human-derived viruses, bovine-derived viruses, and porcine-derived viruses. The types of viruses should be tested comprehensively, and the test methods should meet the requirements. In addition, it is necessary to strengthen the supplier audit of such raw materials, establish a standard quality management system, and establish in-house specification based on the specification of supplier testing and risk assessment.

　　2. Test and Control during the Manufacturing Process

　　The samples at the most appropriate stage in the manufacturing (e.g., unprocessed bulk) are selected for testing and control of adventitious viral agents, and corresponding studies should be carried out to justify the test stage in the manufacturing.

　　For the test methods, the specific requirements are described in "VIII 1. Control of Starting Materials and Raw Materials for Manufacturing".

　　Contents above may be the common control strategies for testing for adventitious viral agents of the aforementioned products. However, in some cases, it is necessary to adopt a control strategy that based on specific product conditions, research and verification results, and combined with or complementary to the following controls: e.g., virus removal/inactivation process steps, release testing (if necessary, viral exogenous agent testing in the drug substance/finished product may also be considered).

IX. Stability Study

　　Stability study is designed to indicate the stability characteristics of intermediate samples or finished products through a series of experiments and provide justification for establishing product shelf life. It also provides the evidence for the manufacturing process, formulation, packaging materials, storage, and transportation condition of drugs and the basis for establishing specification. The stability study can be conducted with reference to the general principles and relevant requirements of Guidance for Stability Study of Biological Products (Interim) and ICH Q5C. Reasonable study protocol should be designed according to the product characteristics, clinical use, storage, packaging, and transportation. The protocol generally includes study category, samples, items, study conditions, study time and study results, etc.

　　The stability study generally includes influencing factor testing, accelerated test and long-term test. Long-term stability study is the main basis for determining the shelf life of the product, and the study duration should cover the proposed shelf life of corresponding intermediate samples or finished products.

　　Study samples generally include drug substance (if applicable), finished product, intermediate requiring temporary storage (if applicable). Stability studies should be conducted using samples manufactured by a representative process and packaged in commercial storage containers or containers that are made from same material and represent worst exposure conditions.

　　The study items and conditions should be determined according to the product characteristics, storage, transportation and clinical use. It is suggested that the study items should cover the test items sensitive to storage/transportation/use conditions, such as titer, purity and impurity, biological activity and other characteristics. These sensitive test items can be based on the accumulation of previous studies, literature reports and research consensus, etc.

　　The setting of study time points should follow the general requirements of Guidance for Stability Study of Biological Products (Interim). Flexible adjustment of test time is optional to accommodate some special characteristics of certain products.

　　The analysis of study results should be performed on different test items respectively, and comprehensive evaluation on the test results of product stability should be conducted.

　　Transportation Stability Studies: Stability studies during simulated and/or actual transportation should be conducted according to the actual shipping environment and conditions of drug substance (if applicable) and drug product.

　　In-use Stability Study: Generally, in-use stability study includes the thawing study of finished product, reconstitution study and compatibility study with dosage devices, etc. In-use stability study should simulate the actual use conditions. Reasonable use conditions and standard operating procedure should be established based on the study results.

X. Package and Container Closure System

　　This chapter includes the containers for drug substance, intermediate samples, and drug product. Since the package and container closure system directly affects the quality of the finished product. When selecting the packaging material, its functionality and safety should be considered to ensure the stable product quality and meet the requirements for clinical use. In general, the package and container closure system should meet the requirements of good tightness, low temperature resistance and label contents should guarantee product’s traceability without any ambiguity. A comprehensive compatibility study and package integrity study should be conducted before marketing application. The study should be conducted using the product manufactured by the representative process and the representative package and container closure system. In study design, the compatibility and integrity of the container and container closure system under special conditions should be considered.

XI. Terms and Definitions

Parental genotype virus:

refers to the starting virus with clear source and used for recombinant virus construction. It may be a wild type virus, an attenuated or adapted virus, or a virus modified by genetic modification technology.

Molecular variants:

refer to the unexpected variants generated during the virus packaging or manufacturing, which may include the variants formed by mutation of certain gene sequences and/or amino acid sequences and may also include the parental genotype virus (or wild type virus) formed by recombination or reverse mutation.

XII. References

　　[1] China Food and Drug Administration. Technical Guidances for Research and Evaluation of Cellular Therapy Products (Interim). 2017.

　　[2] Center for Drug Evaluation. General Principles for Technical Review of Cell Substrates for Vaccine Manufacturing. 2005.

　　[3] Center for Drug Evaluation. Technical Guidances for Stability Studies of Biological Products. 2015.

　　[4] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (2020) [M]. 2020.

　　[5] European Medicines Agency (EMA). ICH Considerations: oncolytic viruses [EB/OL].2009.

　　[6] European Medicines Agency (EMA). Guidance on the quality, non-clinical and clinical aspects of gene therapy medicinal products [EB/OL].2018.

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

　　[8] ICH. Q5C Stability testing of biotechnological and biological products [EB/OL].1995.

　　[9] ICH. Q5D Derivation and characterization of cell substrates used for manufacturing of biotechnological/biological products [EB/OL].1997.

　　[10] ICH. Q5E comparability of biotechnological/biological products subject to changes in their manufacturing process [EB/OL].2004.