Center for Drug Evaluation

　　August 2020

1. Introduction

　　Messenger RNA(mRNA) vaccine is an RNA-based product that was produced by in vitro transcription or synthesis in the presence of DNA template encoding antigen sequence(s) and was delivered into the cells through a specific delivery system. It enables the cells to express the target antigen(s), resulting in specific immune responses for the prevention of infectious diseases.

　　The mRNA vaccines have the following main characteristics: (1) it can enter the cells to express target antigen(s) in vivo, so as to skip the protein expression and purification process in vitro; (2) it can stimulate the immune system to elicit humoral and/or cellular immune responses for corresponding immunoprophylactic and/or immunotherapeutic effects; (3) the delivery system may act as partial adjuvants, in order to enhance immune response or stimulate the immune system to produce a variety of cytokines for modulating the immune responses; (4) mRNAs are degraded by cellular metabolism, reducing mutagenesis risks of gene mutations induced by gene integration.

　　Although mRNA vaccines have made some progress in the clinical research against different pathogens, such as cytomegalovirus (CMV), influenza virus, Ebola virus and Zika virus, numerous issues remain to be further clarified. The issues, which may impact the vaccine efficacy, safety, and product quality, include the potential intrinsic immunogenicity of mRNA, the stability of the delivery systems (e.g., lipid nanoparticles), the safety of the cationic polymers/lipids and the resulted nanoformulations, the delivery efficiency and targeting capability.

　　The delivery system used in an mRNA vaccine is generally composed of a complex formulation. Many special considerations are mandated concerning the CMC, which are: (1) the molecular design of the mRNA, such as the 5’ cap structure, incorporation of untranslated region(UTR), optimization of coding sequence, and chemical modification of nucleotides, etc., may have an impact on the stability of mRNA molecule, protein expression, and immunogenicity of the target antigen; (2) the composition, structure and manufacturing process of drug product are of special concern. The main characteristics or special considerations include the use of cationic polymers or lipid materials, structural diversity of delivery system, particle size control and nanosize-related effect and risk, process complexity, etc. The identification of key attributes during formulation optimization and manufacturing process development are required for the establishment of the control strategy; (3) When novel excipient(s) and/or component(s) with immunomodulatory effects are involved, specific safety studies should be performed.

　　This guideline was drafted for the emergency prevention and control of current COVID-19 outbreak with incomplete scientific knowledge about this type of vaccines. It is intended to guide the development of mRNA vaccines under emergency conditions and to clarify the basic requirements for mRNA vaccine development presently. The content will be updated as more research experience and knowledge of SARS-CoV-2 and the mRNA vaccines become available. This guideline does not imply regulatory recommendations for the COVID-19 vaccine types.

　　This guideline mainly pertains to non-self-amplifying mRNA vaccines. For self-amplifying mRNA vaccines and multivalent/multi-antigen/multicomponent mRNA vaccines, more studies should be conducted based on their special characteristics while referencing this guideline.

　　In view of the incremental and progressive feature of CMC studies in the development of new vaccines during a public health emergency, the applicants should make an overall development plan in advance. The road map of the CMC development, research protocols and risk control strategies in different stages should be submitted and discussed as early as possible. At any stage, if a planned study is modified or omitted, the applicants is required to justify the basis and reasons, and ensure sufficient communication with the regulatory agency before submitting data.

2. Template/Sequence design, plasmid DNA construction and cell bank information

　　2.1 Selection of antigen gene sequence and design of DNA template

　　2.1.1. Rationales and source of antigen selection

　　The antigen source, protein structure, and gene sequences, should be precisely defined, and homologous to the antigen nucleotide and amino acid sequences in current epidemic strains in China should be analyzed; the choice of the target antigen, and the role or action mechanism of the mRNA expression in preventing COVID-19 should be clarified.

　　For SARS-CoV-2, it is recommended to consider the potential effects of antibody-dependent enhancement (ADE) that may be related to different antigen sequences and the risk of pulmonary immunopathological reactions in preclinical toxicology studies. Important characteristics such as the antigen sequence, molecular weight, molecular formula, disulfide bonds (if any) and modifications (if any) should be provided.

　　2.1.2. Sequence design and structure of DNA template

　　(1)The design of the DNA template should be comprehensively described. In addition to the mRNA sequence encoding the target antigen, the design of the functional elements and the data of the study for the verification should also be provided, e.g., design of the cap structure, selection of the promoter, design of 5’ UTR and 3’ UTR, design of the signal peptide, design of the Poly(A) tailing structure/length, type of the nucleoside triphosphate (NTP) used, and its modifications.

　　(2) If any modification, alteration or optimization (e.g., codon optimization) is performed on the mRNA sequence encoding the target antigen, the rationale and purpose of the modification or sequence alteration (e.g., improving mRNA translation efficiency, reducing innate immunogenicity, enhancing stability) should be explained in detail. It is necessary to provide schematics of the structure, based on risk-benefit assessment of genetic modifications or sequence reconstruction, confirmed supportive study results should be provided. If possible, the immunogenicity of the constructed mRNA vaccine itself should be evaluated.

　　In some cases, if the constructed gene sequence includes extra gene sequences other than target antigen, the role and selection basis of these sequences introduced should be analyzed, e.g. to facilitate S-protein trimerization, The corresponding sequence design and supporting data should be provided.

　　2.2 Construction and preparation of DNA template plasmids

　　(1) The sequences and sources of control elements and selection markers of the DNA template should be elaborated, such as promoter, terminator, antibiotic resistance gene. The use of antibiotics should comply with the relevant requirements in the Pharmacopoeia of the People's Republic of China.

　　(2) Detailed information on the construction, preparation, and methods used for identification and verification of the DNA template plasmids should be provided.

　　(3) For DNA template plasmids, whole sequence analysis and validation should be performed combined with verification of the cell banks of the engineered microorganism. Full-length nucleotide sequence of the DNA template used to produce mRNA should be provided, especially for the analysis on control elements of DNA templates, inserted target gene sequence, and variation of selection marker gene.

　　2.3 Construction and testing of cell bank

　　A cell bank system should be established in accordance with the Chinese Pharmacopoeia or with internationally recognized requirements, if the preparation of the DNA template involves the construction of a plasmid and the utilization of an engineered microorganism. Moreover, the test report on the cell bank, issued by the national drug control institution, should be provided.

　　(1) The source, genotype, phenotype of the host microorganism, as well as the selection process of target clones should be clarified. Studies on the verification of host bacterium, such as identity, antibiotic susceptibility, etc., are encouraged.

　　(2) Construction and testing of the engineered microorganism: the qualified plasmid is transformed into an appropriate host microorganism using the optimized transformation process. Subsequently, the cell bank system should be established after the selection of the clone.

　　(3) Cell bank testing: It should be ensured that seed bank is free from contamination by adventitious agents, and that the sequence of target gene and other elements are correct. Testing of the cell bank should include bacterial morphology, culture purity, restriction map of the plasmid, and the sequence of the target gene and other DNA Elements. The testing on cell viability plasmid retention rate, identity, antibiotic resistance, etc., are encouraged.

　　(4) Genetic stability study:

　　Genetic stability analysis (such as DNA sequence, restriction map of plasmid, and plasmid copy number) of the cell bank should be carried out. The passage number should be defined and justified for each level of the seed banks.

　　The size of the cell bank, storage conditions, amplification condition and the passage number allowed should be specified.

3. Manufacture of mRNA vaccines

　　3.1 General considerations

　　During process development, the effects of critical process parameters on the quality attributes of mRNA and/or drug product should be investigated. The manufacturing process and in-process control strategy of mRNA and/or the drug product should be established.

　　The materials to be used in clinical trial should be produced in an appropriate scale, with continuous production and scale-up feasibility. The clinical trial materials should be manufactured under conditions in compliance with GMP.

　　The consistency and controllability of the production process should be comprehensively evaluated considering the development stage, platform prior experience of the process, maturity of the process and adequacy of in-process testing. During product development, data should be continuously accumulated to verify the consistency and controllability of the process. Critical process parameters should be identified prior to the initiation of the commercial production. On the basis of these process parameters, the process control strategy should be established, including the control methods of the critical and primary process parameters, testing of intermediate product, and the test methods validation. Study data should suffice to support the analysis and evaluation of the process stability and batch-to-batch variabilities.

　　3.2 Production of mRNA (drug substance)

　　3.2.1. Raw materials for drug substance production

　　The raw materials used for production should comply with the relevant provisions in the current version of Chinese Pharmacopoeia and/or comply with international recognized requirements.

　　Source, specification and testing reports of raw materials other than the engineered bacterium should be provided. The information on the raw materials listed below should be provided in particular: nucleotides and modified nucleotides; 5’-cap analogues; enzymes (e.g., T7 RNA polymerase, enzymes added during mRNA capping) and buffers used in the in vitro transcription of mRNA; and solvents, and purification media (chromatographic column, magnetic beads, filter membrane) used in the manufacture of drug substance. For raw materials (e.g., T7 RNA polymerase, pyrophosphatase, RNase inhibitor) prepared in-house by recombinant technology or biological/chemical synthesis technology, the corresponding manufacturing process and quality data should be provided. The fidelity of various enzymes used in the production should be analyzed. Specifications for raw materials such as 5’-cap analogues and nucleotides should include purity tests that adequately characterize product-related impurities using mass spectrometry (MS), nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC), etc. In principle, ingredients of human or animal origin should be avoided during the manufacturing of drug substance and drug product. If substances of human or animal origin are used, the risk assessment for adventitious agents should be provided in accordance with the relevant provisions of the Chinese Pharmacopoeia and/or with reference to guidelines such as ICH Q5A.

　　3.2.2. Process development and in-process controls

　　The mRNA drug substance manufacturing process is generally divided into two stages: preparation of DNA templates and preparation of mRNA. Plasmid DNA amplification or PCR amplification, purification and linearization can be used for the preparation of DNA templates. The mRNA preparation process usually includes in vitro transcription using DNA templates, mRNA capping, dephosphorylation, DNase treatment and mRNA purification steps. mRNA modification (e.g., addition of modified nucleosides) and Poly(A) tailing are usually performed during transcription. Capping may be performed during transcription or as a separate step.

　　The production process workflow should be clarified, and a flow chart should be submitted to describe the purpose of the corresponding process, process flow, in-process controls, material flow and intermediate products, etc. The data of each step in the drug substance manufacturing process should be provided, and various process parameters in each step of the manufacturing process should be optimized to establish stable process and corresponding in-process control strategy.

　　Process qualification research information should be submitted, including qualification of in-process control (including whether critical quality attributes of intermediates meet the acceptance criteria, whether critical and key process parameters are within the control range, etc.), batch release data, and necessary impurities removal efficiency data.

　　(1) Preparation of DNA template

　　If the DNA template plasmid amplification/linearization process is used, studies and optimization of the process parameters such as plasmid concentration, linearizing enzyme concentration, incubation time and temperature, should be considered. If the PCR amplification process is used, studies and optimization of the process parameters such as temperature, PCR amplification system, cycle number, time and temperature should be considered. The critical process parameters and their control ranges for the preparation of DNA templates should be qualified and corresponding in-process control specifications such as linearization efficiency, template concentration, sequence accuracy, purity and residual impurity should be established.

　　If a storage of the intermediate is required, the conditions and methods of storage should be determined, and relevant supportive studies should be performed.

　　(2) mRNA production

　　The critical process parameters in the in vitro transcription, Poly(A) tailing, capping, dephosphorylation, DNase treatment and other process steps of mRNA synthesis should be investigated and optimized to qualify the critical process parameters and their acceptable ranges. For example, 1) reaction system, RNA polymerase concentration, NTP concentration, incubation time, temperature, and condition for reaction termination in the in vitro transcription process; 2) DNase concentration, treatment time, temperature, condition for reaction termination, etc. in the DNase treatment process (if any); 3) RNA concentration, temperature and time, capping reaction buffer system, feeding ratio of the material added (e.g., 5’-cap analogues, guanosine triphosphate, ribonuclease inhibitors, related transferases), feeding mode, reaction temperature, time, etc., should be studied in the capping step. During the process, attention should be paid to the efficiency of capping, degradation of mRNA fragments and the accuracy of the sequence. The capping type and the proportion of different capping types should be studied; 4) phosphatase concentration and reaction time in the dephosphorylation process (if applicable); and 5) modified nucleotide, modification type, purification method and purity after modification, feeding ratio, etc. should be clearly defined when the modification of nucleotides are involved.

　　In-process control tests should be performed for above process steps, such as capping efficiency, length of Poly(A) tail, mRNA sequence integrity, sequence accuracy, purity, mRNA concentration, concentration of side-reaction products (incomplete mRNA, double-stranded RNA, truncated RNA, long-stranded RNA, etc.), residual protein, residual DNA, sterility and endotoxin.

　　(3) mRNA purification

　　The purpose of each purification process step during mRNA production should be defined and the impurity profile should be established. Purification method, medium selection, dynamic load, recovery rate, impurity removal rate, etc., should be studied. The critical process parameters of mRNA purification process should be optimized and qualified.

　　Corresponding in-process controls should be established for the mRNA purification process, including the analysis of purified products, such as mRNA concentration, mRNA sequence accuracy and integrity, and removal rate of product- and process-related impurities.

　　(4) Process qualification

　　In addition to the production and release testing of consecutive batches, potential impurities during the mRNA purification process should be investigated. The sources, removal steps and removal capabilities of potential product-related impurities and process-related impurities in mRNA production should be provided, including 5’-uncapped RNA, double-stranded RNA (dsRNA), long-stranded RNA, truncated RNA, residual template DNA, residual enzyme substrate and endotoxin. Safety of the residues should be assessed and, when necessary, toxicological analysis should be performed.

　　3.3 Formulation and manufacturing process (drug product)

　　The formulation and manufacturing process of the drug product should be specified and justified. The source, specifications and test reports of excipients should be provided.

　　The function and content of each component in the drug product formulation should be specified and justified. The candidate formulation should be screened and selected referencing prior delivery system platform knowledge. Evaluations should be made concerning the interaction between mRNA and the delivery materials, the protective effect on the mRNA stability (studies on the degradation of mRNA and mRNA products in the presence of serum or nuclease), mRNA transfection efficiency (endocytosis and endosomal escape), mRNA translation (cell-free extract, cells, etc.), animal pharmacodynamics (immune responses and protective effect), safety evaluation, and manufacturing process consistency.

　　Data (including methods, results and conclusions) should be provided to justify the critical process steps and parameter settings of the drug product, including the data obtained from process development studies evaluating the effects of various manufacturing process parameters on the quality attributes of the drug product, e.g. the complexing efficiency, encapsulation efficiency, particle size and size distribution, drug loading capacity, lipid composition and nitrogen/phosphate ratio (N/P ratio, the molar ratio of protonable amine groups to mRNA phosphate groups). The results consistency with predicted values should be analyzed.

　　For lyophilized product, studies on the effect of lyophilization process on product quality, nanoparticle-related properties, potency before and after lyophilization should be conducted, and appropriate manufacturing process parameters should be established.

　　3.3.1 Raw materials and excipients (adjuvants or delivery systems)

　　Critical materials/excipients (lipids, cationic polymers, etc.) used in the preparation of nanoparticles and delivery systems should be adequately evaluated and quality controlled. In principle, the choice of each component in the delivery system should be justified, and study data on their sources (natural or synthetic, especially sources of lecithin and macromolecules etc.), synthesis processes, quality control characteristics, and stability should be provided, These materials may include but not limited to cationic lipids (DOTAP ((2,3-dioleoyl-propyl) -trimethylamine), DC-Chol (3β- [N- (N',N'-dimethylaminoethane) carbamoyl] cholesterol) and DLin-MC3-DMA, etc.), phospholipids/lipids (DSPC, DOPE, DPPC and cholesterol), PEGylated lipids (mPEG-DSPE, PEG-c-DMA), cationic polymers (polyethyleneimine (PEI), etc.), and their derivatives.

　　(1) Novel excipients such as cationic polymers or cationic lipids may be used in the vaccine delivery system, and their compositions may vary in different drug products. the substance were approved for pharmaceutical use, its chemical composition, specifications, and common usage should be provided including data on completed toxicological safety studies and human safety studies. Given the possible differences in the manufacturer`s processes and impurity profiles, it is recommended that the excipient manufacturers be selected as early as possible based on comprehensive characterization of the material and knowledge on its key quality attributes. In the cases of supplier change or manufacturing process change, it is recommended for the drug developer to conduct adequate studies to ensure that the changes do not negatively affect product quality.

　　If the excipient/delivery substance has not been used in any drug product anywhere, then its functional mechanism, CMC data (including the source, synthesis process, quality control attributes etc.), and safety profiles should be studied in detail according to Guideline for Nonclinical Safety Evaluation of Novel Excipients(NMPA). If PEGylated materials are used, the chemical structure illustration, synthesis process, distribution profile of PEG modification and purity should also be provided.

　　(2) It is recommended to investigate and optimize the robustness of the excipient manufacturing process. Due to the complexity of mRNA complexation and packaging in the delivery system, it is recommended to investigate the delivery system preparation using different batches of polymer materials or lipid composition. The structural integrity and particle size distribution of the nanoparticles resulted should be evaluated to ensure quality consistency and controllability. It is also recommended to set clear specifications for the cationic polymers or novel lipid materials based on the study results. It is recommended for the applicants to clarify the residual impurities and identify key elements that may affect the quality attributes of drug product. The quality specifications should contain purity tests methodology that can adequately characterize product-related impurities, such as MS, NMR, HPLC etc.

　　(3) Adjuvants: In view of the complexity, of adjuvant effects, and the current world-wide development status of the mRNA delivery systems, it is not recommended to add additional adjuvants. If an adjuvant is required, it should be ensured that the added adjuvant does not cause unacceptable toxicity. It is required for the applicant to prove the specific immunomodulatory effect of the adjuvants by clear functional studies before clinical use, and pay attention to the risks that may be introduced concerning the mRNA stability, translation capacity and, unexpected immune reactions. The benefits of adjuvants in enhancing antigen specific immune response must outweigh the risks. A complete set of adjuvant CMC study information should be submitted in accordance with the adjuvant-related guidelines.

　　3.3.2. mRNA encapsulation/loading and Purification

　　mRNA delivery systems may include, but not limited to, lipid-based delivery systems (e.g., lipoplexes), polymer-based delivery systems (e.g., polyplexes), and lipid- and polymer-based delivery systems (e.g., lipopolyplexes). The process of mRNA loading/packaging usually involves the interaction between the packaging materials with the mRNA resulting in the formation of complexed nanoparticles and core-shell structures. Subsequently, the drug product manufacturing process may also include purification steps to remove unencapsulated/unloaded mRNA, un-complexed polymer or lipid material, and/or other substances introduced during the packaging process. The materials and/or compositions used in the delivery systems, the complexation process with mRNA and the formation of nanoparticles may all critically affect the mRNA vaccine quality. The mRNA loading/packaging mechanism, the delivery system optimization criteria, and the complexation and purification process development routes should be all be discussed in detail.

　　The description of mRNA vaccine manufacturing process includes:

　　(1) Complexation of mRNA with delivery system materials;

　　(2) Purification of mRNA nanoparticles.

　　Critical process parameters in the complexation stage may include N/P ratio (ratio of cationic material to mRNA), concentration of each complexation components, buffer system, pH and complexing duration. Both the complexation components and process conditions should be evaluated and optimized. The effects of N/P ratio on the complexation efficiency and mRNA stability should be investigated. Appropriate in-process controls and testing criteria should be proposed for intermediates in the complexation process, e.g., size distribution and polydispersity.

　　Other critical process parameters may also include mixing solvent concentration, flow rate at mixing, mixing pressure, dilution and purification parameters, and sterile filtration conditions. It is recommended to pay attention to the effects of process parameters on the aggregation and/or dissolution of nanoparticles, mRNA leakage, mRNA stability, as well as the relationship between N/P ratio, mRNA stability, transfection efficiency and expression efficiency. Appropriate control criteria should be set for the quality control of nanoparticles, such as mRNA content, encapsulation efficiency, Zeta potential, particle size and size distribution, content of excipients and impurities.

　　3.3.3. Process qualification

　　The qualification and evaluation data of the manufacturing process of consecutive batches should be provided, at least including testing analysis and validation of the quality of intermediates and drug product under the conditions of certain manufacturing process, and the data on the removal of process-related impurities and product-related impurities etc.

4. Characterization

　　Characterization of mRNA vaccines may be referred to relevant guidelines for commercial siRNA lipid nanoparticles and nano-products. Providing the data from routine release test analysis and using advanced analytical technology to conduct quality attributes tests and characterization. Characterization generally includes structural properties (especially those related to the function of mRNA delivery system), purity, impurity (process and product related impurities), in vitro and in vivo potency and immunological properties. In addition to routine release tests, more studies should be considered in the characterization, and it is encouraged to conduct the studies on other structural properties that affect vaccine potency or safety.

　　In the early stage of development, samples should be subjected to preliminary structural confirmation, and the data should be submitted. The complete structural confirmation data can be submitted at the time of filing New Drug Application for marketing approval. The biological potency of vaccines is a comprehensive indicator that reflecting process performance and product quality, therefore relevant studies are recommended to be conducted as early as possible.

　　In the characterization study of delivery system, attention should be given to the representativeness of sampling and the impact of sample preparation procedure on the samples.

　　4.1 Identity and physicochemical properties of mRNA

　　Analysis should be performed on properties such as identity of sequence (including key elements affecting vaccine stability, transcription, translation and expression efficiency), mRNA concentration (ultraviolet absorption), mRNA modification ratio, capping efficiency, integrity, purity, physical properties (e.g. appearance, pH, etc.), in vitro mRNA translation capacity should be analyzed. If possible, the intrinsic immunogenicity of the constructed mRNA vaccine should be evaluated.

　　4.2 Structural and physicochemical properties of nanoparticles

　　The critical quality attributes of the mRNA delivery nanoparticle should be determined by impacts on product quality and potency. It is recommended to evaluate the effects of mRNA loading capacity, pH, complexation and/or encapsulation efficiency, and resulted mean particle size and distribution, particle structure shape, Zeta potential, as well as the mRNA leak/release mechanisms. The mRNA delivery systems need to be characterized concerning the phospholipid composition, N/P ratio, loaded and unloaded mRNA content, un-complexed polymer and lipid, and delivery material related impurities (such as oxidation/degradation products of lipids containing unsaturated double bonds, and/or synthetic polymer byproducts). If applicable, analysis of nanoparticle aggregates and PEG modification structure are recommended. The correlations between the delivery nanoparticle quality attributes (e.g., particle size and distribution, surface charge, encapsulation efficiency) and the resulted immunization potency needs to be discussed. In addition, mRNA release mechanism is also thought to be closely related to its efficacy. It is encouraged for the applicant to conduct studies on the release characteristics of mRNA and set up in vitro release assay for the evaluation of drug release under lysosomal pH conditions.

　　Pertaining to the mRNA-delivery system interactions in the preparation of mRNA vaccine drug products, it is recommended to conduct comprehensive studies on the correlation of physicochemical and/or structural properties, including the isoelectric point and, pKa of the delivery materials, particle size and distribution of the resulted delivery nanoparticle, particle morphology, encapsulation efficiency location of mRNA, in the nanoparticles, and mRNA leakage or release rates with the resulted biological activities, including mRNA delivery, antigen expression, immune responses and toxicities.

　　4.3 Impurities

　　Potential impurities may include process and product related impurities that were generated during the manufacturing process, storage, and/or related to the sealed containers used, and/or found in the stability study batches. For IND applications, list of impurities should be submitted and discussed according to their sources and safety, risks, including but not limited to, the mRNA related impurities, residual DNA, residual host-cell proteins, delivery materials related impurities, uncomplexed delivery materials and manufacturing process related impurities. It is recommended to consider toxicology study results, in combination with literature data, previously accumulated knowledge and information, etc. Major impurities need to be monitored and analyzed, and when necessary, included in the quality specifications and safety evaluation plans. For later phase clinical trials, in addition to the information provided in the early clinical trial application, further analysis, isolation, identification of the impurities are required. It is recommended to justify the inclusion or exclusion of in-process controls or release criteria concerning any impurity considering its correlation with vaccine efficacy and safety., For items included in the quality control system, the acceptable criteria should be optimized with the progression of the research. For compendial tests, the standards in the Pharmacopoeia must be met.

　　For mRNA related impurities, it is recommended to pay attention to truncated sequences affecting mRNA function (possibly resulting from incomplete transcription or degradation/breakage of mRNA), double-stranded mRNA sequences that may result in nonspecific immune responses, incompletely capped mRNAs, cap-related impurities, incomplete dephosphorylated mRNAs, over-modified mRNAs, etc. In addition, attention should be paid to mRNA mismatch sequences, mRNA oxidation products, etc.

　　The presence of residual DNA unlike in the cases in other conventional vaccines, is a residue encoding specific DNA sequences, so the safety of residual DNA should be evaluated considering the encoded sequence, residual amount, fragment size, etc.

　　It is also recommended to pay attention to: (1) delivery material-related impurities, including impurities introduced during material synthesis and impurities generated during the complexing with mRNA; (2) oxidation and related degradation products of unsaturated lipids; (3) particles resulting from nanoparticle aggregation; and (4) uncomplexed delivery materials; unencapsulated mRNAs; and mRNAs that may be degraded or inactivated during manufacturing and storage. Among these, uncomplexed delivery molecules could affect the stability of nanoparticles; and free mRNA would be easily degraded, both leading to poor efficacy.

　　4.4 Bioassays

　　In vivo potency assay: According to mechanism of mRNA vaccines, antigen-specific humoral and/or cellular immunity should be evaluated as the biological activity. Neutralizing antibody and/or total antibody assays may be used for the evaluation of humoral immune responses. Whenever possible, animal models that are closely related to the human immune system should be selected. For establishing the assays of neutralizing antibody and/or total antibody titers, the selections of virus (pseudovirus) strains for neutralization, antigens for antibody capture, and the reference material etc. need to be studies. When necessary and possible, it is encouraged to develop or implement methods to define the antibody responses including IgG subtype characterizations and neutralizing epitope analysis, etc. It is also suggested to evaluate cellular immune responses (e.g., Elispot assay for CTL response detection). These measurements can at least be used as a corresponding quality specification to support CMC change evaluations during clinical trials.

　　In vitro assay: Transfection of mammalian cells in vitro and measurements of the expressed antigen(s) are generally required. It is suggested for the applicant to establish a method for quantitative detection of the expressed antigen(s). The criteria for antigen(s) expression, the sensitivity of the method, and the accuracy of the quantification should be validated. The expression profile needs to be characterized, and the molecular weight of expressed antigen(s) should match the predicted molecular weight(s). It is encouraged to establish corresponding methods with validation for each expressed antigen and predicted profile specifications. It is encouraged to conduct correlation studies between the in vitro assays of immunogenicity and efficacy in animal models

　　Animal challenge-protection study: it is considered as one of the most ideal pre-clinical evaluation methods for efficacy and it can be conducted in combination with pharmacodynamic studies.

　　Activity of co-expressed gene sequences: In case that the mRNA sequence also includes cytokine sequences with functions of regulating immune response or sequences with adjuvant effects, in addition to the COVID-19 antigen sequence, it is recommended that detailed analysis of such molecules be conducted, including the molecular identity, expression level and immunological effects. If this cytokine or the sequence with adjuvant effects have not been used in any marketed drug product, then separate pharmacology and toxicology studies should be conducted for such molecules.

5. Specifications

　　For clinical trial application, the specifications of the mRNA candidate vaccine candidate can be preliminarily established based on the data collected during the process development and qualification. In the stage of commercial production, complete specifications should be in place based on the results of risk control analysis and process validation performed according to the relevant guidelines. The tests described in this section are recommended to be performed. During the early development stage, these tests can be used as in-process controls for data accumulation. Whether these tests should be included in specifications for commercial manufacturing can be decided based on the data accumulated during clinical trials. For general process-related impurities, if it has been fully validated that these impurities can be removed effectively and consistently, and therefore well controlled in the process, the tests of these impurities may be excluded from the release test.

　　5.1 DNA template

　　It is recommended to consider the following tests for quality control: identity, concentration/content of DNA template, sequencing, purity, linearization efficiency (if applicable), residual impurities, bioburden, endotoxin, etc. The applicants are encouraged to establish quality control methods for the in vitro transcriptional activity of DNA templates.

　　The residual impurities in the DNA template may include residual host cell DNA, host cell RNA, host cell protein, etc.

　　mRNA sequencing is less accurate than DNA sequencing and is limited by its transcriptional length, therefore sequencing of DNA templates is essential to ensure the accuracy of mRNA sequence.

　　5.2 mRNA drug substance

　　It is recommended to consider the following tests for quality control of the mRNA drug substance: mRNA identity, length of mRNA, integrity and accuracy of mRNA sequence, mRNA physicochemical characteristics (e.g., pH, appearance, etc.), quantity of mRNA, capping efficiency, purity, product-related impurities (e.g., incomplete mRNA, double-stranded RNA, etc.), process-related impurities (e.g., residual enzyme, residual DNA template, residual organic solvents, residual metal ions, etc.), sterility, endotoxin, etc.

　　5.3 Intermediates

　　Based on the actual manufacturing process of mRNA vaccine drug product, critical intermediates should be defined and intermediate specifications should be established. Such intermediates may include the complexes of the mRNA and delivery system materials, nanoparticle intermediates, etc., the testing of intermediates is also a part of in-process control. The following factors should be considered for the definition of intermediates and to establishments of corresponding test requirements: (1) whether the stage is the most sensitive stage of corresponding test item; (2) whether there is an impact on the active ingredient from subsequent manufacturing process and formulation of drug product, such as whether lyophilization is performed; and (3) whether the subsequent process steps should be tested at this step, e.g., the active ingredient content is used to guide compounding.

　　It is recommended to consider the following quality control tests: physical characteristics, identity, content, endotoxin, sterility, etc.

　　(1) Physical characteristics: including pH, appearance, nanoparticle size, PDI, and Zeta potential, etc.

　　(2) Identity: it should be confirmed by appropriate methods, such as sequencing, electrophoresis, HPLC, etc.

　　(3) Content: including concentration of nucleic acid, percentage of encapsulation, which can be determined by appropriate methods such as UV absorption at 260nm or fluorescence analysis.

　　(4) Residual process-related impurities.

　　(5) Endotoxin and sterility tests.

　　5.4 Drug product

　　It is recommended to include the following tests for quality control: product identity and mRNA sequence confirmation, contents (mRNA, delivery system materials and related excipients), physicochemical characteristics of the mRNA delivery system, purity, related impurities (residues), bioassay, safety tests, etc.

　　5.4.1. Identity

　　mRNA and associated delivery system components should be identified by appropriate methods.

　　5.4.2. Content

　　Quantity of mRNA, mRNA integrity, mRNA purity, quantity of delivery system components, adjuvant content (if applicable), and the content of other special excipients (if applicable) should be determined.

　　5.4.3. Physicochemical properties

　　The physicochemical properties of the mRNA delivery system, which may impact the safety as well as the efficacy of the drug product, should be defined, including but not limited to the nanoparticle size distribution, Polydispersity index (PDI), Zeta potential, pH, etc. In addition, it is also recommended to include general quality control measures of drug products, such as appearance, filling volume/filling volume accuracy, visible particulates, residual moisture (if applicable), etc.

　　5.4.4. Purity, process-related impurities and residues

　　The mRNA encapsulation efficiency, the residual of solvents (e.g., ethanol) and other process-related impurities should be quantified and included in the quality tests. It is recommended to establish appropriate purity specifications, develop appropriate methods for the detection of oxidation/degradation products of the lipids (e.g., DOPE), and analyze the impacts of impurities on the safety and efficacy of the drug product.

　　5.4.5. Bioassays

　　It is recommended to include in vitro and/or in vivo bioassays as quality control tests of the drug product. In the early stage of development, it is recommended for the applicant to develop appropriate in vivo potency bioassay and set up criteria based on preliminary pharmaceutical research. When considered necessary based on the mechanism of vaccine, quality control tests for cellular immune responses should be established. Considering the variability of bioassays, it is recommended to use the ratio between vaccine product and some reference vaccine and set the product specification.

　　5.4.6. Safety tests

　　Safety tests generally include endotoxin, abnormal toxicity test, sterility, etc.

　　5.5 Analytical method and validation of the analytical methods

　　Factors include types of the test methods selected, pretreatment of the sample (e.g., reverse transcription, enrichment, enzyme digestion, lysis, etc.), testing conditions, etc., will affect the reliability of the test results for mRNA drug substance and drug product. Therefore, the test methods should be qualified as required, multiple methods should be used to analyze the important indicators such as mRNA purity, capping efficiency, particle size distribution of the lipid nanoparticles, and results should be obtained by methods based on different principles so that they can be validated mutually. Appropriate test methods for quality control should be selected based on the validation results in method sensitivity, accuracy, precision and robustness.

　　Validation data of the tests, provided in the IND submission, should suffice to preliminarily verify the applicability of these test methods. Validation data provided of tests for important quality indicators or critical quality attributes (e.g., percentage of encapsulation, capping efficiency, Zeta potential, particle size and distribution, purity, in vitro assay, in vivo potency, etc.) should be consistent with or applicable for quality control and importance of the method in early development. Complete method validation data should be provided in accordance with relevant guidelines by the time of application for marketing authorization.

　　5.6 Reference materials

　　Data on the source, preparation, testing results, calibration and stability studies (periodic retest) of established reference materials or reference standards (including being used to measure the nucleic acid content, purity, in vitro assay, in vivo potency, complete sequence determination) should be provided in clinical trial application.

6. Stability studies

　　The stability studies and evaluation of mRNA vaccines should be conducted in accordance with the relevant guidelines on stability evaluation of biological products.

　　Stability-indicating parameters should be defined and selected for stability testing with a focus on the physicochemical characteristics and expression efficiency of mRNA, such as percentage of encapsulation, quantification of active substance, particle size and distribution, Zeta potential, aggregation and in vivo potency of nanoparticles, supplemented by pH, appearance, and bioburden/sterility. Stability should be investigated under following conditions: temperature changes, pH changes, light exposure (photostability), high humidity (for lyophilized mRNA), repeated freezing-thawing (when stored frozen), after reconstitution or in use, etc.

　　If any intermediate needs to be stored during the manufacturing process of the vaccine, stability assessments or relevant validation studies of the intermediate should also be conducted, the condition and method for the intermediate storage should be specified, and their usability for production should be studied.

7. Containers

　　For drug product, container-closure system compatibility study should be conducted in accordance with relevant guidelines. For all consumables (e.g., storage bags, silicone tubing, microfluidic chips, pipelines) used in drug substance and drug product manufacturing process that directly contact with the product, compatibility data or other applicable supporting data should be submitted.

8. Special considerations regarding CMC development and changes during development under emergency conditions

　　In emergency situations, in order to accelerate vaccine development and provide better guidance for the application dossier, it is recommended to consider the following strategies in emergency situations: accumulating development data, sample testing, in-process control and release control in the early stage of product development; and timely identifying and correcting deficiencies on the basis of risk identification. Nevertheless, CMC studies need to be gradually improved during the clinical trial in accordance with the requirements for the marketing authorization of conventional vaccines. Timely communications and follow-up will be conducted during the clinical trial, so as to collect on-site feedback information and adjust the regulatory review strategies according to the epidemic situation.

　　The preliminary considerations proposed for the manufacturing process, quality characterization and specifications in early CMC development:

　　8.1 Cell banks

　　Attention should be given particularly to scientific issues including adventitious agents, DNA sequence consistency, and also on the selection of gene sequences, which can be demonstrated with the results of toxicological and pharmacodynamic studies of the product.

　　8.2 Manufacturing process

　　Applicants with prior knowledge of the platform may use the knowledge, after preliminary verification, to develop the manufacturing process and establish the process controls of the COVID-19 vaccine in the early stage, and continue to scale up and optimize the process parameters during clinical trials. However, it needs to be ensured that critical steps of the manufacturing process, which may affect product safety, are fully validated. Performance indicators for in-process controls are recommended to be established as many as possible for accumulation of product knowledge and process knowledge, in order to lay a foundation for comparability studies, in case problems show up during process scale-up. The reduction of control indicators should be considered after sufficient data accumulation and process validation.

　　The formulation and manufacturing process of drug product can be proposed based on the prior knowledge of the platform, and the preclinical pharmacodynamic and toxicology data. After the preliminary verification of vaccine stability, antigen-adjuvant/delivery system interactions should be investigated in depth during clinical trial to further optimize the formulation of drug product. The process scale-up of lipid nanoparticle product may be accompanied by changes in process equipment. Attention should be paid to the continuous optimization and adjustment of process parameters to ensure the quality comparability of lipid nanoparticles. Continuous validation (cleaning, sterility assurance, usage times, etc.) of manufacturing equipment at different scales and the compatibility study should be conducted.

　　Manufacturing scale should at least be able to support early clinical studies. The consistency and controllability of the manufacturing process should be verified by producing at least 3 consecutive batches, which should be completed before initial clinical trials.

　　8.3 Characterization

　　The structure of the mRNA vaccine product should be characterized and the data should be provided in the early stage of development, while the complete data of structural characterization should be provided in the New Drug Application. Studies related to the biological potency of the vaccine, which is a comprehensive evaluation of process performance and product quality, are recommended to be carried out as early as possible.

　　8.4 Specifications

　　Since production batches, scale and quality control methods are in the initial stage of the development, the comprehensiveness of test items should be considered. For example, test items should include purity, process-related impurities, product-related impurities, biological activity, etc. It is encouraged to accumulate adequate and comprehensive product quality data during clinical trials. Preliminary investigation of method suitability, and validation data of the test methods for important indicators or critical quality attributes (e.g., protective efficacy), which is consistent with the control and the importance degree of the developmental phase should be provided, while the confirmation of product-related impurities and comprehensive method validation can be carried out during clinical trials. The specification limits can be preliminarily established or reported based on the results of preclinical toxicology studies, process scale-up, stability studies, etc., which can be determined by the accumulation of more manufacture experience during the clinical trials. For quality control indicators related to product safety (e.g., microbial contamination control indicators, hazardous substance residue), it is recommended to conduct methodology validation as early as possible, with a minimum requirement to include the verification of suitability.

　　The in vivo potency, as an important reference for predicting the product effectiveness in human, is one of Critical Quality Attributes (CQA) of a vaccine product, therefore, it should generally be performed for drug product. Other test items, such as mRNA content, mainly reflect the effect of antigen on the potency of drug product, therefore, should be carried out. The feasibility of replacing in vivo potency with in vitro assay can be evaluated post marketing authorization, decisions will be made based on in vivo and in vitro assay data from sufficient batches and the comparative analysis of commercial batches and clinical batches.

　　8.5 Stability data supporting the conduct of clinical trials should be provided in the clinical application stage

　　If available, alternative or other supporting information (e.g., using the same packaging materials, excipients, formulation as that of marketed vaccines) should be submitted.

　　8.6 Manufacturing changes during development

　　CMC changes are often unavoidable during drug development, especially in the early stage of development. Preclinical pharmacological and toxicological studies using process representative batches of clinical trial samples are encouraged. In case of changes from preclinical to clinical batches, detailed comparative information before and after the change should be provided, and the post-change process should be described and analyzed, and risks should be assessed in detail. Corresponding comparability data should be provided to demonstrate that the changes do not adversely affect product quality.

　　During the clinical trials, continuous changes such as production scale-up and process optimization may be accompanied, so adequate comparability studies should be conducted to assess the potential impact of the changes on product quality. The comparability studies should be designed in advance, and the sampling batches, steps and tests items to be performed should be planned in advance, with particular attention to the representative retention samples at each developmental stage. In addition, a comprehensive study of the reference materials for key indicators such as antigen content and animal potency are beneficial to ensure the quality of the product and the traceability of the reference materials.

　　If quality comparability analysis is inadequate to support that the change has no undesirable impact on the product, it should be necessary to supplement nonclinical or even clinical data, such as immunogenicity comparison and necessary safety comparison. In view of the complexity and current limited knowledge of mRNA vaccines, for major changes during clinical trials, it is recommended to perform comparability analysis and comprehensive studies on potency, including humoral immune responses and cellular immune responses before and after the change.

9. Terminology

mRNA vaccines:

Messenger RNA(mRNA) vaccine is an RNA-based product that was produced by in vitro transcription or synthesis in the presence of DNA template encoding antigen sequence(s) and was delivered into the cells through a specific delivery system. It enables the cells to express the target antigen(s), resulting in specific immune responses for the prevention of infectious diseases.

Delivery system:

a deliver system often condenses, complexes and/or packages the nucleic acid drug with specific delivery material(s) to increase the stability of the nucleic acid drug and protect it from nuclease(s) and/or other degradation mechanisms. It facilitates the transportation of nucleic acid drug through the cell membrane by interacting with cells, and enables intracellular delivery of the drug into cytoplasm (for siRNAs and mRNA) or nucleus (for DNA) hence to express target proteins.

Multivalent/Multi-antigen/Multicomponent mRNA vaccines:

mRNA vaccines containing the target gene sequences of different SARS-CoV-2 antigens that are constructed separately, but co-packaged in the same delivery system.

10. References

　　1. Miao Hefan, Guo Yong, Jiang Xinxiang, et al. Research Progress and Challenge of mRNA Vaccines [J]. Journal of Immunology, 2016, 32 (05): 446-449.

　　2. Lin Man, Zeng Xiancheng, Hong Min, et al. Research Progress in mRNA Vaccines [J]. Journal of Cellular and Molecular Immunology, 2013,29 (6): 658-660.

　　3. Schmid A. Considerations for Producing mRNA Vaccines for Clinical Trials[J]. Methods Mol Biol, 2017, 1499:237-251.

　　4. Hinz T, Kallen K, Britten C M, et al. The European Regulatory Environment of RNA-Based Vaccines[J]. Methods Mol Biol, 2017, 1499:203-222.

　　5. FDA. Guidance for Industry-Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. CBER. November 2007.

　　6. Kauffman K J, Webber M J, Anderson D G. Materials for Non-viral intracellular delivery of messenger RNA therapeutics[J]. J Control Release,2016, 240:227-234.

　　7. FDA. Guidance for Industry-Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation. CDER. April 2018.

　　8. FDA. Guidance for Industry-Drug Products, Including Biological Products, that Contain Nanomaterials (draft guidance). CDER&CBER. December 2017.