Guidance on Submission of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Clinical Trial Applications

This guideline addresses the specific documentation requirements for evaluation of 

Chemistry, Manufacturing, and Control Information parts in Investigational New 

Drug Applications (INDs) submitted to the Saudi Food and Drug Authority.

The requirements defined in this guideline are sourced from the following guidelines:

- The FDA Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), and

- The EMA Guideline on the requirements for quality documentation concerning biological investigational medicinal products in clinical trials.

The objective of this document is to address the quality requirements of an investigational medicinal product for a given clinical trial and not to provide guidance on a Company's overall development strategy for a medicinal product. 

​​Gene therapy products are biological products, administered for the prevention, treatment, or cure of a disease or condition of human beings. According to the SFDA Guideline on Classification of Advanced Therapy Medicinal Products, a gene therapy medicinal product mediates its effect by expressing the recombinant nucleic acid or its product. An example gene therapy product would be ex vivo genetically modified human cells. 

Clinical trial application, including clinical trials for gene therapy products, is required to describe the CMC information for the investigational product including drug substance (DS) and the Drug Product (DP).  SFDA may place the product on clinical hold if the clinical trial application does not contain sufficient CMC information to assess the risks to subjects in the proposed studies. 

The CMC information submitted in a clinical trial application describes the applicant's commitment to perform manufacturing and testing of the investigational product as stated.  It is acknowledged that manufacturing changes may be necessary as product development proceeds, and the applicant should submit information amendments to supplement the initial information submitted for the CMC processes.  In addition, if manufacturing is changed, the applicant should submit the change for SFDA review prior to implementation. The Saudi Clinical Trial Registry SCTR published a guidance for submission on the following link: https://sctr.sfda.gov.sa/Guidance.aspx?lang=ar 

SFDA recommends that applicants begin to organize and categorize their CMC information in an investigational medicinal product dossier (IMPD), according to the eCTD (electronic common technical document format) for Module 3.

The amount of CMC information to be submitted in a clinical trial application depends on the phase of investigation and the scope of the clinical investigation proposed. The emphasis for CMC review in all phases of development is product safety and manufacturing controls.  It is expected that applicants may need to make modifications and additions to previously submitted information as clinical development proceeds and additional product knowledge and manufacturing experience is collected. 

 The following sections provide detailed recommendations for submitting IMPD.​​

​1. Administrative documents

Administrative documents (e.g., application forms, cover letters, reviewer guides), and labeling information should be submitted. The cover letter should include a brief explanation of the submission and its contents. When amendments are submitted to the clinical trial application for manufacturing changes, the cover letter should clearly describe the purpose of the amendment and highlight proposed changes. 

2. Labels

The clinical trial application, must contain a copy of all labels and labeling to be provided to each investigator in the clinical study.  It is recommended to include sample or mock-up labels in the administrative section of the application.  The applicants should note that investigational products must bear a label indicating that it is for “investigational use only”. Other important labeling information such as those 

- For products derived from autologous sources the statement: “FOR AUTOLOGOUS USE ONLY”, should be used. And, 

- If donor testing and screening is not performed, the product should be labeled “NOT EVALUATED FOR INFECTIOUS SUBSTANCES”

3. Previously Submitted Information

Should the applicant wish to cross reference another product, the applicant is required to submit a written statement in the clinical trial application that appropriately identifies information previously submitted. The applicant should describe the information and identify where that information is located in the previously submitted file.​

1. General information

The Clinical trial application, should contain a general introduction to the quality aspects product (safety, identity, purity, potency/strength and stability) of the investigational product, mode of action, and proposed clinical use. Description of potential Critical quality attributes (CQAs) that are relevant to the safety and biological activity of the product as they are understood by the applicant at the time of submission should be provided. A CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.  SFDA acknowledges that as the applicant’s knowledge about the product is increased through development stages. In the early clinical trial phases, understanding of products characteristics/CQAs is limited and as the clinical trials progress, understanding of the products is expected to advance, therefore the identities of CQAs and their limits should be refined.  It is also recommended during early stages, to conduct extensive product characterization to help the applicant identify and understand CQAs, which in turn will help in defining manufacturing process controls, manufacturing consistency, and stability.

2. Drug Substance and Drug Product

The clinical trial application must contain a description of the Drug substance (DS) and Drugs product (DP), including the physical, chemical, or biological characteristics, manufacturing controls, and testing information, to ensure the DS and DP meet acceptable limits (safety, identity, purity, potency/strength and stability)

Drug substance (DS) means the same as “active pharmaceutical ingredient” which is further described as any substance that is intended for incorporation into a finished DP and is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body. 

Drug product (DP) is defined as “a finished dosage form that contains an active drug ingredient generally, but not necessarily, in association with inactive ingredients. Additionally, a vector in its final formulation used for administration of the genetic material may also be an active ingredient, depending upon the manufacturing process and formulation of the finished dosage form.

It is recognized that distinguishing a DS from a DP may be difficult for some gene therapy products due to the complex nature of the manufacturing processes.  Some gene therapy products may not have a distinct DS.  Others may consist of two or more different DSs that are combined to make the DP.  When the manufacturing process includes more than one DS, it is recommended to provide separate DS sections for each active ingredient of the final product. A summary of the available stability data for the DS and the DP, recommended storage conditions, and shelf life, if applicable, should also be included in each DS and DP section.  

3. Combination Products  

For submissions in which the gene therapy is a component of a combination product, it is recommended to briefly describe the combination product in the summary and briefly state the regulatory status of each component. 

4. Product Handling at the Clinical Site  

Proper control of the finished DP is critical to the investigational studies.  Therefore, the clinical trial application should include a description of how the product will be shipped to, received, and handled at the clinical site to ensure safety, product quality, and stability.  The clinical trial application should also include information on shipping conditions, storage conditions, expiration date/time (if applicable), and chain of custody from the manufacturer to the site of administration. Information for product handling at the clinical site prior to administration (such as thawing, the addition of diluent or adjuvant, loading into a delivery device, and transport to the bedside) and summary information on product stability prior to and during administration (e.g., in-device hold times and temperatures) should be provided. Details regarding product stability after preparation for delivery and delivery device compatibility data should be included in IMPD (sections 3.2.P.8 and 3.2.P.2.6) respectively.  Instructions for drug handing and preparation for administration at the clinical site (e.g., Pharmacy Manual) should be provided in the appropriate section of the clinical trial application. 

​​ 1.1. General Information  

a. Nomenclature

The applicant should provide the name of the DS(s).  If the name of the DS has changed during clinical development, the applicant should provide the names used to identify the DS at all stages of development. 

b. Structure

The applicant should submit information on the molecular structure (including genetic sequence, if applicable) and/or cellular components of the DS.  The genetic sequence can be represented in a schematic diagram that includes a map of relevant regulatory elements (e.g., promoter/enhancer, introns, poly(A) signal), restriction enzyme (RE) sites, and functional components (e.g., transgene, selection markers).  The applicant should also submit information on the sequence analysis and the annotated sequence data in the application.  It is recommended that the sequence data, including any sequence data collected to support the genetic stability of the vector, also be submitted.  

Some specific examples of structure and structural elements of different gene therapy products are outlined below: 

- For viral-based gene therapies, the applicant should include a description of the composition of the viral capsid and envelope structures, as appropriate, and any modifications to these structures (e.g., modifications to antibody binding sites or tropism-changing elements).  It is recommended to include biophysical characteristics (e.g., molecular weight, particle size) and biochemical characteristics (e.g., glycosylation sites).  The applicant should also describe the nature of the genome of viral vectors, whether single-stranded, double-stranded, or self-complementary, deoxyribonucleic acid (DNA) or RNA, and the copy number of genomes per particle.   

- For microbial-based gene therapies, the applicant should include defining physical and biochemical properties, growth characteristics, genetic markers (e.g., auxotrophic or attenuating mutations, antibiotic resistance), and the location (e.g., on plasmid, episome, or chromosome) and description of any inserted foreign genes and regulatory elements, if applicable.  

- For ex vivo genetically modified cell-based gene therapies, such as Chimeric Antigen Receptor modified T cells (CARTs), the applicant should describe the expected major and minor cell populations, as well as the vector that contains the transgene cassette that is transferred into the cell.  For cells that have been genetically modified using genome editing, the applicant should describe the gene(s) that is altered and how the change(s) was made (i.e., the gene editing technology used). For vectors used in ex vivo modification of cells, information should be submitted as a regulatory support file (RSF). 

c. General Properties

The applicant should provide a section in the clinical trial application that describes the composition and properties of the DS, including the biological activity and proposed mechanisms of action.  

1.2. Drug Substance Manufacture 

a. Manufacturer(s)  

The applicant must provide the name and address of each manufacturer, including contract manufacturer(s), involved in the manufacture, testing, and storage of the DS.  You should indicate the responsibility of each manufacturer. Your clinical trial application should contain complete information on the DS manufacturer, regardless of whether the process is performed by you or by a contract manufacturing organization (CMO).

b. Description of Manufacturing Process and Process Controls​

The description of the DS manufacturing process and process controls should include the following, as applicable:  cell culture; transduction; cell expansion; harvest(s); purification; filling; and storage and shipping conditions.  The description should also accurately represent the process and process controls.  It is acknowledged that information on process controls may be limited early in development and recommend that applicants provide additional information and updates as product development proceeds.  Changes and updates to the DS manufacturing process and process controls information should be submitted as an amendment to the clinical trial application prior to implementation for investigational use in clinical studies. 

- Batch and Scale 

A description of how applicant defines each manufacturing run (i.e., batch, lot, other) should be submitted with an explanation of the batch numbering system.  The applicant should clearly state whether any pooling of harvests or intermediates occurs during manufacturing.  If pooling is necessary during production, we recommend that you control the storage conditions (e.g., time, temperature, container closure system) for each pool and that you describe the testing performed in the clinical trial application.

It is also recommended that the applicant provide an explanation for how the batch scale is defined (e.g., bioreactor volume, cell processing capacity) and how the DS is quantified (e.g., vector genomes, transducing units, infectious particles, mass, number of gene modified cells) to facilitate review and allow a better understanding of the manufacturing process. When known, please include the yield expected per batch.

- Manufacturing Process 

The description of the manufacturing process should include a process flow diagram(s) and a detailed narrative.  The description should clearly identify any process controls and in-process testing (e.g., titer, bioburden, viability, impurities) as well as acceptable operating parameters (e.g., process times, temperature ranges, cell passage number, pH, CO2, dissolved O2, glucose level).  It is acknowledged that this information may be gathered over the course of product development and may be submitted in a stage appropriate manner.     

We recommend that you monitor process performance parameters for process consistency. Process trend analysis and evaluation of process parameters and materials will help to determine and establish process control strategies. You should clearly describe any controls for cleaning and change over as well as tracking and segregation procedures that are in place to prevent cross- contamination throughout the manufacturing process.

o Cell Culture (for Vector Production) 

The description of all cell culture conditions should contain sufficient detail to make understandable any of the process steps that apply, process timing, culture conditions, hold times and transfer steps, and materials used (e.g., media components, bags/flasks).  

You should describe whether the cell culture system is open or closed and any aseptic processing steps. If extensive culture times are needed, you should outline the in-process controls you have in place to monitor cell quality (e.g., viability, bioburden, pH, dissolved O2).

o Vector Production 

For the manufacture of gene therapy vectors (e.g., virus, bacteria, plasmids), the applicant should provide a description of all production and purification procedures.  

Production procedures should include the cell culture and expansion steps, transfection or infection procedures, harvest steps, hold times, vector purification (e.g., density gradient centrifugation, column purification), concentration or buffer exchange steps, and the reagents/components used during these processes. You should outline any in-process testing to ensure vector quality as appropriate (e.g., titer, impurities). 

The applicant should describe whether the vector DS will be formulated into the DP for administration of the genetic material or whether it will be formulated as a bulk DS for ex vivo genetic modification of cells.  As an active pharmaceutical ingredient, an appropriate level of control should be applied to each DS, and each DS should be manufactured under current good manufacturing practice (CGMP) conditions that are appropriate for the stage of development.  

o Genetically Modified Cell Production 

If the gene therapy product consists of genetically modified cells, cell processing description should contain sufficient detail to make understandable any of the following process steps that apply: source material (e.g., autologous or allogeneic cells; donor eligibility when applicable); collection of cellular source material (e.g., leukapheresis, biopsy); storage at the collection site; shipping to and handling at the manufacturing facility; cell selection, isolation, or enrichment steps (including methods, devices, reagents); cell expansion conditions; hold times and transfer steps;  cell harvest and purification, if any, and materials used.  

The applicant should also provide a complete description of all procedures used for gene modification (such as transfection, infection or electroporation of vectors, or genome editing components) and any additional culture, cell selection, or treatments after modification.  The vector used should be described in detail as indicated above. 

o Irradiated Cells 

If the product contains or is processed with irradiated cells, the applicant should provide information on the irradiator source, documentation for the calibration of the irradiator source and provide supporting data to demonstrate that the irradiated cells are rendered replication-incompetent, while still maintaining their desired characteristics. 

o Filling, Storage, and Transportation (Shipping) 

The applicant should provide a detailed description and identify any associated process controls for formulation, filling, storage, and shipping of the DS, if applicable.  The applicant should also describe the container closure system used for storage and shipping of the DS.  It is recommended that the applicant describe procedures that are in place to ensure appropriate storage and transport (as needed). 

c. Control of Materials 

The applicant must provide a list of all materials used in manufacturing and a description of the quality or grade of these materials.  This information includes the identity of the material, the supplier, the quality (such as clinical-grade) the source of material (e.g., animal, human, insect), and the stage at which each material is used in the manufacturing process (e.g., cell culture, vector purification).  This includes information on components, such as cells, cell and viral banking systems, and reagents, as described in more detail below; it also includes raw materials and equipment that come into contact with the product, such as culture bags, culture flasks, chromatography matrices, and tubing.  It is important to note that information relevant to materials and their control should be provided under the section control of materials in the drug substance and drug product as appropriate.   

The applicant should establish a risk-based materials qualification program and provide documentation that the materials used for manufacturing meet standards appropriate for their intended use (e.g., test results, certificates of analysis (COAs), package inserts).  The nature and extent of materials qualification, including the testing required, will depend on the specific material and its risk potential, the manner in which it is used in the manufacturing process, and the stage of clinical development. 

- Reagents 

For the purposes of this guidance, reagents (or ancillary materials) are those materials used in the manufacturing process, but that are not intended to be part of the final product. Examples include fetal bovine serum, enzymes (e.g., trypsin, collagenase, DNase/RNase, restriction endonucleases), growth factors, cytokines, monoclonal antibodies, antibody-coated beads, antibiotics, culture media and media components, and detergents. These reagents can affect the safety, potency, and purity of the final product, especially by introducing adventitious agents or other impurities. 

o Biological materials

Animal-derived materials increase the risk of introducing adventitious agents. Certain animal-derived materials, such as sera, are complex mixtures that are difficult to standardize, and such materials may have significant batch-to-batch variations that may affect the reproducibility of your manufacturing process or the quality of your final product. We recommend that you consider using non-animal-derived reagents if possible (e.g., serum-free tissue culture media and recombinant proteases).  Note that a recombinant reagent can still present adventitious agent-related risks, depending on how the reagent is manufactured.  

For biological materials such as material derived from animal or human sources, SFDA may require information on the manufacturing and/or testing to evaluate the safety and quality of the material.  For example: 

• Bovine derived materials: Information on the source of the material, location where the herd was born, raised, and slaughtered; and any other information relevant to the risk of transmissible spongiform encephalopathy (TSE).  

If serum is used, SFDA recommends that it be γ-irradiated to reduce the risk of adventitious agents. This information may be included on the COA and Certificate of Origin (COO) provided from the supplier.

• Recombinant proteins (such as cytokines): 

Qualification of recombinant proteins should be risk-based.  SFDA-approved pharmaceutical-grade recombinant proteins do not require further adventitious agent testing, but may require testing to demonstrate performance in manufacturing the gene therapy product.  

Other recombinant proteins may require targeted adventitious agent testing, depending the cell lines and materials used in their production and purification.  The applicant should consider that many recombinant proteins (such as cytokines) and monoclonal antibodies used during the manufacture of gene therapy products may be purified by affinity chromatography using antibodies generated from mouse hybridomas. This may introduce the risk of contamination with adventitious agents from rodents, which should be controlled by the supplier.

• Murine or Monoclonal Antibodies:  

The applicant should determine and, if necessary, address the risk of contamination with adventitious agents from rodents. Alternatively, if a Master File for the antibody has been submitted to SFDA, the applicant should provide a Letter of Authorization to cross-reference this information. 

• Human Source: applicant should specify if the reagent used is clinical or research grade and provide a COA or information regarding testing of the donor or reagent. 

a. Human albumin

It is recommended to use SFDA approved products, and to have procedures in place to ensure that no recalled lots were used during manufacture or preparation of the product.

b. Human serum

Human AB serum may be used in manufacturing the gene therapy product.  However, SFDA recommends that the applicant ensure the AB serum used in manufacturing the product does not have the potential to transmit infectious disease.  If the serum is derived from Source Plasma, which may be pooled from a large number of donors and is not tested as extensively as blood products intended for infusion, the applicant may reduce the risk of infectious disease transmission by conducting additional testing for relevant transfusion-transmitted infections.  Alternatively, it may be acceptable to include viral inactivation or clearance steps in the production of human AB serum from Source Plasma.  

c. Other human-derived reagents

For all other reagents that are human-derived, the applicant should identify whether the reagent is a licensed product (e.g., HSA, IL-2) or is clinical or research grade and provide a COA or information regarding testing of the donor or reagent.

d. Cells - Autologous and Allogeneic Cells or Tissue

For autologous or allogeneic cells or tissue, the applicant should provide a description of the cell source, the collection procedure, and any related handling, culturing, processing, storage, shipping, and testing that is performed prior to use in manufacture.  The description should include the following information:

- Materials used for collection (including devices, reagents, tubing, and containers).

- Method of cell collection (i.e., standard blood draw, bone marrow aspiration, or apheresis).

- Enrichment steps, cryopreservation, if performed.

- Labeling and tracking of collected samples.

- Hold times

- Transportation conditions to the manufacturing facility

As an example, for cells collected by leukapheresis:  the applicant should provide a summary description of the collection device(s); operating parameters; volumes or number of cells to be collected; and how the collected material is labeled, stored, tracked, and transported to the manufacturing facility.  Establishing well designed process controls and standard operating procedures (SOPs) for manipulating and handling starting and in-process materials can help reduce variability in the manufacturing process and ultimately in the DS and DP.   

This is especially important for multi-center clinical trials, where establishing standardized procedures for cell collection and handling across all collection sites is critical to assuring the quality and safety of the final product as well as ensuring control of the manufacturing process.  The clinical trial application should include a list of collection sites complying with SFDA regulations.  

a. Autologous Cells 

No donor eligibility screening is required for cells and tissues for autologous use. However, the applicant should determine, based on patient information, whether the manufacturing procedures could increase the risk to the patient by further propagation of pathogenic agents that may be present in the starting material (patient cellular material).  This may require additional information.  

SFDA recommend that the applicant describe precautions to prevent the spread of viruses or other adventitious agents to persons other than the autologous recipient and cross contamination with another patient’s material. 

b. Allogeneic Cells 

Allogeneic leukapheresis starting material does require a donor eligibility determination, including screening and testing for relevant communicable disease agents and diseases.

Allogeneic cells from a single donor or source tissue may sometimes be expanded and stored for greater consistency and control in manufacturing. In these situations, SFDA generally recommends qualifying allogeneic master and working cell banks in the same way as cell banks used for production of viral vectors (see Cell Banking Systems, below), provided that sufficient cellular material is available for this testing. In these situations, SFDA is chiefly concerned about the introduction of adventitious agents (e.g., viruses, bacteria, and mycoplasma) during the bank manufacturing process, especially from human or bovine materials, animal feeder cells, other animal-derived reagents, or human AB serum, if used. If the allogeneic cell bank is small, and limited material is available for testing, the applicant should consult with the Quality Reviewer of the application for more information on appropriate cell bank qualification.

o Cell Banking System

A banking system improves control and consistency in the manufacturing of many biologics. Banking assures an adequate supply of equivalent, well-characterized material for production throughout development and commercialization. For these reasons, banked materials are a common starting point for many routine production applications. 

It is recommended that the applicant provide a description of the testing of the cell bank and relevant COA(s) in section 3.2.A.2 of the IMPD.  Information on bank qualification and adventitious agent testing should also be included in the comprehensive “Adventitious Agents Safety Evaluation” (3.2.A.2) section of the IMPD. 

d. Control of Critical Steps and Intermediates 

The applicant should describe the control of critical steps and intermediates in the manufacturing process. It is recommended that the applicant also consider any steps in which in-process tests with acceptance criteria are performed as critical control steps.  SFDA acknowledges that this information may be limited in the early phases of development and recommends that applicants provide additional information and updates as product development proceeds.   

The applicant should define manufacturing intermediates and provide information on the quality and control of intermediates.  Intermediates may include material from collection or hold steps, such as temporary storage of bulk harvest, concentration steps, or purification intermediates (e.g., column fractions or eluate).  The duration of production steps and hold times should be controlled and recorded. 

It is recommended that the applicant provide information on the plasmid manufacturing procedures, reagents, and plasmid specifications for use, regardless of whether they were made by the applicant or a contract manufacturer.  In general, it is recommended that this testing include assays to ensure the identity, purity, potency, and safety of the final product.  For a DNA plasmid, this may include sterility, endotoxin, purity (including percent of supercoiled form and residual cell DNA, RNA, and protein levels), and identity testing (restriction digest and sequencing if sequencing was not performed on the bacterial bank).  A COA documenting plasmid quality testing should be included in the application.

e. Process Validation and/or Evaluation 

SFDA does not require process validation studies for early-stage manufacturing (i.e., Phase I-II).  It is recommended that the applicant use early-stage manufacturing experience to evaluate the need for process improvements and to support process validation studies in the future. At all stages of development, however, the manufacturer must have established written SOPs to ensure proper manufacturing control and oversight.  

f. Manufacturing Process Development  

The applicant should provide a description and discussion of the developmental history of the manufacturing process. For later stage of phases, there may be changes to the manufacturing process as part of process development, optimization, or under certain conditions there may be reprocessing step(s).  It is recommended that the applicant describe how manufacturing differences are expected to impact product safety and activity in the “Batch Analysis” section. 

If the applicant makes significant manufacturing changes, then comparability studies may be necessary to determine the impact of these changes on the identity, purity, potency, and safety of the product.  The extent of comparability testing would depend on the manufacturing change, the ability of analytical methods to detect changes in the product, and the stage of clinical development.  For first-in-human studies, any differences between toxicology lots and clinical lots should be assessed for their impact on product safety.  For later phase studies, especially those designed to measure product efficacy, differences in clinical lots should be assessed for their impact on product safety and activity.  The comparability plan should be discussed with SFDA prior to beginning comparability studies.   

SFDA strongly recommends the applicant to retain samples of the DS and manufacturing intermediates, when possible, in case comparability studies are required in future.

1.3. Drug Substance Characterization

a. Elucidation of Structure and Other Characteristics

It is recommended that the applicant include annotated sequence analysis for the vector in the application and any additional sequence information gathered during the course of product development in subsequent submissions. In case of ex vivo genetically modified cells such as CAR T Cell products where the DS contains (patient’s cells and vector), SFDA recommend the applicant to provide the raw sequence data or vector information whether by the applicant or contractor in the regulatory support file.

b. Impurities

It is recommended that the manufacturing process be designed to remove process- and product-related impurities and that the applicant have tests in place to measure levels of residual impurities.  

As the applicant optimizes manufacturing process the impurity profile including acceptance limits must be refined. 

- Process-Related Impurities 

Specifications should be set for materials used in vector production, unless process validation data have been provided to demonstrate that such residues are consistently reduced to acceptable levels.

For the release specifications, tests should be developed and relevant (upper) limits set to monitor the residual levels of contaminants. This include, but are not limited to, residual cell substrate proteins, extraneous nucleic acid sequences, helper virus contaminants (i.e., infectious virus, viral DNA, viral proteins), and reagents used during manufacture, such as cytokines, growth factors, antibodies, selection beads, serum, and solvents, as well as raw materials that may have been used during the production process such as resins or benzonase. 

Common process-related impurities for viral preparations are listed in the table below. 

- Product-Related Impurities 

For viral vectors, typical product-related impurities may include defective interfering particles, non-infectious particles, empty capsid particles, or replicating recombinant virus contaminants.  These impurities should be measured and may be reported as a ratio, for example, (full: empty particles) or (virus particles: infectious units).  

For ex vivo genetically modified cells, product-related impurities include non-target cells, which may be present after selection or enrichment, and unmodified target cells, which may be present after the ex vivo modification step.  It is recommended that the applicant evaluate the nature and number of non-target cells and measure the percentage of cells that have been genetically modified.  As the applicant develop a greater understanding of the cellular phenotypes present in the product during clinical development, the applicant may also consider adding impurity tests for specific cell populations in order to establish greater manufacturing control.   

1.4. Control of Drug Substance 

a. Specifications

The applicant should list DS specifications in the application.  Specifications are defined as a list of tests, references to analytical procedures, and appropriate acceptance criteria used to assess quality.  Acceptance criteria should be established and justified, based on data obtained from lots used in preclinical and/or clinical studies, data from lots used for demonstration of manufacturing consistency, data from stability studies, and relevant development data. 

For products in the early stages of clinical development, very few specifications are finalized, and some tests may still be under development.  However, the testing plan submitted in the application should be adequate to describe the physical, chemical, or biological characteristics of the DS necessary to ensure that the DS meets acceptable limits for identity, strength (potency), quality, and purity.  

For later phases, the applicant should describe assays to assess product characteristics, product safety and purity. To ensure product characteristics the applicant should perfume identity testing e.g. quantitative polymerase chain reaction (qPCR), Safety testing, at the stage of production at which contamination is most likely to be detected.  For example, tests for mycoplasma or adventitious viruses (in vivo or in vitro) should be performed. 

The applicant should also ensure consistent dosing and potency in the clinical investigations, assays used to determine dose (e.g., vector genome titer by quantitative polymerase chain reaction (qPCR), transducing units, plaque-forming units, and flow cytometry for transduced cells) should be qualified as suitable for use prior to initiating clinical studies.  

The applicant should note that the same types of tests listed in this section of the guidance may not be necessary for the release of both the DS and DP.  In certain situations, the DS and DP may not be readily distinguishable due to the design of manufacturing process and control.  In this case, it is recommended that the applicant provide a rationale to support the selection of testing performed for release of either the DS or DP.

b. Analytical Procedures

The applicant should provide a description of the analytical procedures used during manufacturing to assess the manufacturing process and product quality.  In the original clinical trial application submission, the descriptions should have sufficient detail, including a description of system suitability control, so that it can be understood and evaluated for the adequacy of the procedures.  

Non-compendial (in house testing) analytical procedures may be acceptable if the applicant provides adequate information about test method, including specificity, sensitivity, and robustness.  

- Replication Competent Virus 

For non-replicating gene therapy viral vectors, specific testing is recommended, due to the potential for these vectors to recombine or revert to a parental or wild-type (WT) phenotype at a low frequency.  Tests for replication-competent, parental, or wild-type viruses that may be generated during production (e.g., replication competent adenovirus (RCA) and replication-competent retrovirus (RCR)) should be performed on material collected at the appropriate stage of the manufacturing process.  For example, testing banked material for the presence of replication competent viruses is recommended and as a specification for in-process or release testing of the DS or DP, as appropriate. 

o Replication-Competent Retrovirus (RCR) Testing 

Most retroviral-based products (including lentivirus and foamy virus-based products) used for gene therapy applications are designed to be replication defective.  To ensure the absence of RCR, the applicant should perform testing for RCR at multiple points during production of a retroviral vector.  

o Replication-Competent Adenovirus (RCA) Testing 

Most adenoviral-based products used for gene therapy applications are designed to be replication defective.  RCA may be generated at a low frequency as a result of homologous recombination between viral vector sequences and viral sequences present in the cell substrate during manufacturing.  Therefore, for adenoviral-based gene therapy products, it is recommended that the applicant qualify the MVB for RCA and test either the DS or DP of each production lot for RCA.  A maximum level of 1 RCA in 1 RCA in 3 x 1010 viral particles is recommended.  

o Replication-Competent AAV (rcAAV) Testing 

Preparations of AAV vectors can be contaminated with helper virus-dependent rcAAV, also referred to as wild-type AAV or pseudo wild-type AAV.  These rcAAV are generated through homologous or non-homologous recombination events between AAV elements present on the vector and AAV rep and cap sequences that are present during manufacture.  While wild-type AAV has no known associated pathology and cannot replicate without helper virus, expression of cap or rep genes in infected cells can result in unintended immune responses, which can reduce effectiveness and may have unintended safety risks.  Therefore, it is recommended that the applicant test for rcAAV, which could potentially replicate in the presence of helper virus, and report these results in the application.  

o Wild-Type Oncolytic Virus Testing 

Most oncolytic viruses used in gene therapy applications not only carry transgenes but also have been attenuated or adapted from a parental virus strain to grow selectively in cancer cells.  It may be possible for these attenuated or adapted viruses to either recombine or revert to a parental (or WT) genotype during manufacture.  Therefore, it is recommended that the applicant conduct tests to determine whether the parental virus sequences are present in the product.  In addition, it is recommended that the applicant select production cells that do not contain viral sequences that may allow homologous recombination with the product.  For example, 293 cell substrates is not recommended for the manufacture of E1-modified oncolytic adenoviruses due to the potential for homologous recombination with E1 sequences in the 293 cells.

c. Validation of Analytical Procedures

Validation of analytical procedures is usually not required for Phase 1 studies; however, the applicant should demonstrate that test methods are appropriately controlled.  In general, scientifically sound principles for assay performance should be applied (i.e., tests should be specific, sensitive, and reproducible and include appropriate controls or standards).  It is recommended that the applicant use compendial methods when appropriate and qualify safety-related tests prior to initiation of clinical trials.  

To ensure safety of gene therapy products, the applicant should also qualify the assays used to determine dose (e.g., vector genome titer by qPCR, transducing units, plaque forming units, and transduced cells) prior to initiating clinical studies.  The applicant should also provide a detailed description of the qualification protocol (e.g., samples; standards; positive/negative controls; reference lots; and controls evaluated, such as operators, reagents, equipment, dates) and data supporting the accuracy, reproducibility, sensitivity, and specificity of the method.

d. Batch Analysis

The applicant should include a table with test results for batches (or lots) of the DS that they have been manufactured.  For early stage, this may include toxicology lots, developmental batches, engineering runs, or a single manufacturing run for clinical grade material.  It is recommended that the applicant gain adequate experience with a new clinical manufacturing processes prior to making clinical material.  This is especially critical following technology transfer to a new manufacturing facility, when manufacturing changes occur during development, and when multiple manufacturing facilities will be utilized.  It should be noted that batches manufactured in different ways should be clearly identified in the submission. It is recommended that the applicant annually update this section of the application as new batches are produced.  The applicant should indicate any batches that fail to meet release specifications and any action taken to investigate the failure. 

e. Justification of Specification

The applicant should provide justification for the DS specifications in the application.  It is recognized that acceptance criteria may be adjusted throughout the product development stages, based on both manufacturing and clinical experience.  For early stage clinical studies, assays used to characterize production lots may be more variable than those used in later phase investigations. 

For later stage investigational studies in which the primary objective is to gather meaningful data about product efficacy, it is recommended that acceptance criteria be tightened to ensure batches are well-defined and consistently manufactured. 

1.5. Reference Standards or Materials 

The applicant should provide information on the reference standards or reference materials used for testing the DS in the application.  It is recommended that the applicant provide the source and lot number; expiration date; COAs, when available; and/or internally or externally generated evidence of identity and purity for each reference standard.  

Three types of reference standards are generally used:  1) certified reference standards (e.g., USP compendial standards); 2) commercially supplied reference standards obtained from a reputable commercial source; and/or 3) other materials of documented purity, custom-synthesized by an analytical laboratory or other noncommercial establishment.  

In some cases, the reference material for an assay will be a well-characterized lot of the gene therapy product, itself, which is considered an in-house reference standard. In this case, it is recommended that the applicant reserve and maintain a sufficient amount of material (e.g., part of a production lot) to serve as a reference material.

1.6. Container Closure System

The applicant should describe the type(s) of container and closure used for the DS, including the identity of materials used in the construction of the container closure system.  It is recommended that the applicant determine whether the containers and closures are compatible with the DS.  Compatibility with a gene therapy product may be evaluated during stability studies or may be based on historical data and experience, using similar products.

1.7. Stability (3.2.S.7) 

a. Stability Summary and Conclusions

It is recommended that the applicant describe in application the types of stability studies (either conducted or planned) to demonstrate that the DS is within acceptable limits.  The protocol should describe the storage container, formulation, storage conditions, testing frequency, and specifications (i.e., test methodologies and acceptance criteria).  The applicant should note that stability studies may evolve with product development, and if the DS is immediately processed into a DP, long term DS stability data may not be needed.  

The stability analysis may include measures of product sterility (or container integrity), identity, purity, quality, and activity or potency.  It is recommended that the applicant provide justification for the test methods and acceptance criteria used in the stability analysis.  It is often helpful to demonstrate that at least one or more of the test methods in the stability analysis are stability-indicating.  The applicant may demonstrate a test is stability indicating using forced degradation studies, accelerated stability studies, or another type of experimental system that demonstrates product deterioration.

b. Post-Approval Stability Protocol and Stability Commitment

As the applicant progress with product development, considering stability studies that would be required to determine an expiry date might be needed. 

c. Stability Data

It is recommended that the applicant provide the results of the stability studies and update this information on a regular basis (e.g., annual reports) once available. Information on the qualification of analytical procedures used to generate stability data should be submitted.​

2.1. Drug Product Description and Composition (3.2.P.1) 

The applicant should provide a description of the DP and its composition.  This includes a description of the dosage form and a list of all of its components (active and inactive), the amount per unit basis, the function, and a reference to quality standard for each component (e.g., compendial monograph or manufacturers’ specifications).   

2.2. Pharmaceutical Development (3.2.P.2) 

The Pharmaceutical Development section should contain information on the development studies conducted to establish that product formulation, manufacturing process, container closure system and microbiological attributes.  The studies described here are distinguished from routine control tests conducted, according to specifications.  Additionally, this section should identify and describe the formulation and process attributes (critical parameters) that can influence batch reproducibility, product performance, and DP quality.  It is understandable that only limited information may be available at early stages of development; thus, SFDA recommends that the applicant provides more information and updates as the product progresses into late stage of development.

a. Components of the Drug Product (3.2.P.2.1) 

- Drug Substance (3.2.P.2.1.1) 

The applicant should describe the compatibility of the DS with the components listed in the “Description and Composition of the Drug Product” and the key characteristics of the DS (e.g., concentration, viability, aggregation state, viral infectivity) that can influence the performance of the DP.

- Excipients (3.2.P.2.1.2) 

The applicant should describe in the application the choice of excipients and inactive components of the DP listed in the “Description and Composition of the Drug Product”, their concentration, and the characteristics of these excipients that can influence DP performance. 

b. Drug Product (3.2.P.2.2)   

- Formulation Development (3.2.P.2.2.1) 

The applicant should briefly describe the development of the DP formulation, taking into consideration the proposed route of administration and usage in the application.  

SFDA recommends to describe any other formulations that have been used in clinical or preclinical studies and provide a reference to such studies, if applicable.  If formulation changes were needed for stability, device compatibility, or safety concerns, this information can be reported here.  

- Physicochemical and Biologic Properties (3.2.P.2.2.3) 

The applicant should describe the parameters relevant to the performance of the DP, or reference relevant DS sections, as appropriate.  

These parameters include physicochemical such as particle number and size and/or biological properties of the product such as potency and dosing unit. 

c. Manufacturing Process Development (3.2.P.2.3) 

The applicant should describe the selection and optimization of the DP manufacturing process (described in the “Description of Manufacturing Process and Process Controls”) if development studies have been performed.  It is understandable that only limited information may be available at early stages of development; thus, SFDA recommends that the applicant provide more information and updates as the product progresses into late stage of development.  

d. Container Closure System (3.2.P.2.4) 

The applicant should describe the suitability of the container closure system, for the storage, transportation (shipping), and use of the DP. 

SFDA recommends to consider choice of materials, protection from moisture, gases, and light, compatibility with the formulation (including adsorption to the container and leaching), safety of materials, and performance. 

e. Microbiological Attributes (3.2.P.2.5) 

The applicant should describe the final product microbial testing, and address how the integrity of the container closure system to prevent microbial contamination will be assessed.  

For ex vivo genetically, modified cells that are administered immediately after manufacturing, it is recommended a negative test result from an in-process sterility test (on a sample taken 48 to 72 hours prior to final harvest) for release of the DP.  

SFDA will generally consider the submission of other innovative testing such rapid microbiological testing, if scientifically justified.

f. Compatibility (3.2.P.2.6) 

The applicant should discuss the compatibility of the DP with the diluent used for reconstitution or the delivery device, if applicable. 

2.3. Manufacture (3.2.P.3) 

a. Manufacturers (3.2.P.3.1) 

The applicant should provide the name, address, and responsibility of each manufacturer, including contractor manufacturer(s), involved in the manufacture and testing of the DP.  

For gene therapy-device combination products, it is recommended that the applicant list the manufacturing facilities for the device components and describe the assembly and testing processes taking place at each site. 

b. Batch Formula (3.2.P.3.2) 

The applicant should provide a batch formula that includes a list of all components of the dosage form, the amount range per batch as appropriate, and a reference to their quality standards. 

c. Description of Manufacturing Process and Process Controls (3.2.P.3.3) 

The applicant should provide a detailed description of the DP manufacturing process and identify process controls and intermediate tests.  The description should include both flow diagram(s) and narrative description(s) as well as packaging, product contact materials, and equipment used.  This process can include manufacturing steps, such as final formulation, filtration, filling and freezing, and process controls.  Under certain conditions, repeating sterile filtration (if applicable) may be critical.  

SFDA recommends that the applicant clearly describe the conditions and justifications for each reprocessing procedure and demonstrate product consistency between reprocessed lots and normal production lots.   

d. Controls of Critical Steps and Intermediates (3.2.P.3.4) 

The applicant should describe the control of critical steps and intermediates in the manufacturing process.  Critical steps should include those outlined in the “Description of Manufacturing Process and Process Controls” to ensure control as well as steps in which tests with acceptance criteria are performed.  It is recommended that the applicant provide justification for acceptance criteria or limits set for these tests.  In addition, the applicant should provide information on the quality and control of intermediates of the manufacturing process.  Manufacturing intermediates are defined by the manufacturer and may include material from collection steps or hold steps. 

It is understandable that only limited information may be available at early stages of development; thus, SFDA recommends that the applicant provide more information and updates as the product progresses into late stage of development

e. Process Validation and/or Evaluation (3.2.P.3.5) 

SFDA does not require full process validation during clinical development. However, based on the development stage of the product, SFDA may require the applicant to provide information to support the manufacturing process consistency.

2.4. Control of Excipients (3.2.P.4) 

An excipient is any component, other than to the active ingredient that is intended to be part of the final product (e.g., human serum albumin, Dimethyl Sulfoxide (DMSO)). The applicant should provide the following sections:

• Specifications for all excipients. If the excipient is compendial, information may not be required. 

• Analytical procedures for testing excipients.  If the applicant is performing any additional testing or qualification of compendial excipients, the applicant should describe that testing here. 

• Process validation is not expected during clinical development. SFDA recommends that the applicant provide any available validation or verification information for the analytical procedures used to test excipients. 

• The applicant should provide justification for the proposed excipient specifications, as appropriate. 

• For excipients of human or animal origin, the applicant should provide information regarding source, specifications, description of testing performed, and viral safety data.  If human serum is used, it is recommended that the applicant submit information documenting donor suitability as well as appropriate infectious disease testing.  

• For excipients used for the first time in a DP or used for the first time in a route of administration (Novel Excipients), the applicant should provide full details of manufacture, characterization, and controls, with cross-references to supporting safety data (nonclinical and/or clinical) in a regulatory file submitted to SFDA, if available. 

2.5. Control of Drug Product (3.2.P.5) 

a. Specifications (3.2.P.5.1) 

The applicant should list DP specifications in the original clinical trial application submission.  The testing plan should be adequate to describe the physical, chemical, or biological characteristics of the DP necessary to ensure that the DP meets acceptable limits for identity, strength (potency), quality, and purity.  Product lots that fail to meet specifications should not be used in the clinical investigation without providing protocol of out of specifications and SFDA approval. For early phase clinical studies, it is recommended that assays be in place to assess safety and dose of the product.  

It is recommended that product release assays be performed at the manufacturing step at which they are necessary and appropriate. SFDA also recommends to establish or, in some cases, tighten acceptance criteria, based on manufacturing experience as clinical development proceeds. 

b. Analytical Procedures (3.2.P.5.2)

The applicant should describe the analytical procedures used for testing the DP.  If the analytical procedures are the same as those for the DS, the applicant do not need to repeat this information unless there is a matrix effect from the DP on assay performance.  Additional comments regarding analytical tests are provided below: 

- Sterility

It is recognized that the compendial sterility tests (USP <71>; 610.12) may not be suitable for all products (e.g., those with limited shelf life).  

Rapid sterility tests may be acceptable for ex-vivo genetically modified cells such as CAR T Cell products administered fresh or with limited hold time between final formulation and patient administration.  

 The applicant should report the sterility failure to both the clinical investigator and SFDA.  It is recommended that the applicant include results of the investigation of the cause of the sterility failure and any corrective actions in an information amendment submitted to the application after initial receipt of the positive culture test result. 

In some cases, certain product may interfere with the results of sterility testing.  For example, a product component or manufacturing impurities (e.g., antibiotics) may have mycotoxic or anti-bacterial properties.  Therefore, it is recommended that the applicant assess the validity of the sterility assay using the bacteriostasis and fungistasis testing, as described in USP <71> Sterility Tests.   

If the DP is frozen before use, it is recommended that the applicant perform sterility testing on a sample of the product prior to cryopreservation so that results will be available before the product is administered to a patient.  However, if the product undergoes manipulation after thawing (e.g., washing, culturing), particularly if procedures are performed in an open system, the applicant may need to perform additional release testing including sterility and identity testing to ensure product quality.  

It is recommended that the applicant incorporate the results of in-process sterility testing into the acceptance criteria for final product specifications. 

- Identity 

This test is performed on the final labeled product to verify its contents.  SFDA recommends using different types of test methods as a single test is not sufficient to distinguish clearly among products.     

If the final product is ex-vivo genetically modified cells such as such as CAR T Cell products, it is recommended that identity testing include an assay to measure the presence of vector or genetic change and an assay specific for the cellular composition of the final product. 

- Purity 

Product purity is the extent to which the final product is free from any additional substance, regardless of whether they are harmful to the recipient or damaging to the product itself. 

Purity testing includes assays for residual manufacturing impurities of the DS, which may include, but are not limited to host cell proteins and DNA, reagents used during manufacture, such as cytokines and growth factors. 

In the case of ex-vivo genetically modified cells such as CAR T Cell products, any unintended cellular populations should be reported.  

Although the rabbit pyrogen test method is the current required method for testing certain licensed biological products for pyrogenic substances, alternative test methods are generally accepted, such as the Limulus Amebocyte Lysate (LAL).  

- Potency 

The applicant should describe and justify in the application all assays used to measure potency.  A potency assay is not required to initiate early phase clinical studies. 

- Viability  

The applicant should establish minimum release criteria for viability, where appropriate.  For ex-vivo genetically modified cells Such as CAR T Cell products, it is recommended to submit data in support of a lower viability specification, demonstrating, for example, that dead cells and cell debris do not affect the safe administration of the product and/or the therapeutic effect. 

- Cell Number or Dose 

The dose-determining assay is an important part of the DP specifications and should be qualified as suitable for use prior to initiating clinical studies.  If the final product is ex-vivo genetically modified cells Such as CAR T Cell products, the applicant should have an acceptance criterion for the minimum number of genetically modified cells in a product lot. 

c. Validation of Analytical Procedures (3.2.P.5.3)  

SFDA does not require full Process validation during clinical development. However, based on the development stage of the product, SFDA may require the applicant to provide information to support the manufacturing process consistency. Also, it is recommended to provide information to support the qualification of certain safety related or dose-related assays. 

If they are the same as those listed for DS testing, the applicant does not need to repeat them but should reference that section of the application. 

d. Batch Analyses (3.2.P.5.4) 

The applicant should provide final product COA(s), if available, or a description of the batches generated to date, and the results of any batch analyses for the DP used in toxicology studies, engineering runs, or clinical studies. Applicants can also include supportive developmental batches, if appropriate. 

e. Characterization of Impurities (3.2.P.5.5) 

The applicant should provide information on characterization of impurities if not previously provided in the “Impurities drug substance section”. 

f. Justification of Specifications (3.2.P.5.6) 

The applicant should provide justification for the DP specifications

2.6. Reference Standards or Materials (3.2.P.6) 

The applicant should provide information on the reference standards or reference materials used in testing the DP if not previously provided in the “Reference Standards or Materials drug substance section”

2.7. Container Closure System (3.2.P.7) 

The applicant should provide a description of the container closure systems, including identity of construction materials for each primary packaging component (primary packaging is defined packaging in direct contact with the product itself) and its specification.  The applicant should also provide information on how the container is sterilized.  

2.8. Stability (3.2.P.8) 

a. Stability Summary and Conclusion (3.2.P.8.1) 

Summary table of all batches used in stability studies should be listed along with corresponding protocols and results based on that stage of development or clinical trial phase. 

Stability protocols may differ depending on the product type and hold manufacturing time. Long-term stability plan should be submitted as part of the protocol. 

Summary should include storage conditions and shelf life as well as in-use storage conditions.

b. Stability Data (3.2.P.8.3) 

The applicant should provide results of the stability studies in the application in an appropriate format (e.g., tabular, graphic, narrative).  Information on the analytical procedures used to generate the data should also be included. ​

3.1. Adventitious Agents Safety Evaluation (3.2.A.2) 

The applicant is recommended to assess the risk potential contamination with adventitious agents as follows:

• For non-viral adventitious agents (such as transmissible spongiform encephalopathy agents, bacteria, mycoplasma, and fungi).  Submission of certification and or testing of components and control of the production process. 

• For viral adventitious agents, viral safety studies should submit. Study reports and data to support qualification of the manufacturing components (such as adventitious agents test reports for banked materials) may be submitted as a part of this appendix. ​​

Information that is specific to a regulatory region can be put in the regional section of the application.  This would include environmental risk assessment, commitments, protocol, and other information as requested.​

CAR T cells products are regulated as a gene therapy (GT) product under SFDA's existing framework for biological products. This Annex of the guidance addresses general considerations for CAR T product manufacturing and testing and is not designed to be a stand-alone CMC guidance. Please refer to Guidance on Submission of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Clinical Trial Applications.

We recommend applicant organize information in the Common Technical Document (CTD) format with the vector CMC information described in a complete Drug Substance (DS) section.

For CAR T cells in the early stages of clinical development, very few specifications are finalized, and some tests may still be under development. Characterization data collected during early studies can inform release criteria used in later development to ensure product and process consistency. Thus, characterization studies are crucial to support product development and comparability assessments. For studies in which a primary objective is to gather meaningful data about product efficacy, we recommend that acceptance criteria be refined to ensure batches are well defined and consistently manufactured. ​

Vector recommendations (viral/plasmids/bacteria/non-viral methods)

A critical part for manufacturing of CARTs is the vector. A vector is a vehicle used to deliver genetic material. Vectors, whether a virus, bacteria or plasmid have been genetically modified to transfer genetic material. 

The quality of final CART products is greatly affected by the quality of viral vector. SFDA recommends applicants to follow eCTD module 3 format to submit vector information including manufacturing, characterization and analytical testing. This guidance provides specific recommendations and additional details for CAR T cell manufacturing and testing.

Applicants are required to submit information on the manufacturing and testing of the viral vector. 

Manufacturing and testing considerations

Release of viral vector should be based on specifications that ensure:

• Identity: to ensure genomic integrity, this includes structure, vector design, and genetic development information, as well as general characteristics

• Biological activity: this includes both transgene activity and transduction efficiency, which can be linked to final CART cell potency profile. While transgene activity is measured by the release of biological markers, vector performance ability is amount of vector added to produce the required transduction efficiency, while maintaining a targeted safe vector copy number. 

• Purity: applicants are advised to include testing of residual amount of any raw materials used such as benzonase. Other expected impurities should be tested such as residual host cell DNA or RNA and host cell proteins. 

• Safety: this includes sterility, mycoplasma, and endotoxin testing. Adventitious agent testing is different based on type of vector used, as well as the replication competent virus assay as appropriate. 

• Stability: studies to be conducted to establish storage conditions and expiration time of the vector.

Starting material 

Cellular starting material such as leukapheresis must be well characterized. Autologous leukapheresis starting material does not require a donor eligibility determination but you may consider a risk-based approach for screening or testing. However, Allogeneic leukapheresis starting material does require a donor eligibility determination, including screening and testing for relevant communicable disease agents and diseases. 

Collection, handling, and stability

Collection, handling, storage (whether fresh or cryopreserved), as well transportation from collection centers to manufacturing sites should be well documented. 

Stability of starting material during storage and shipment must be demonstrated using shipping validation studies. The studies should provide information on shipping container and temperature control measures such as data loggers.

Cellular material variability

Cellular starting material variability between donors is the main cause of CART cell product lot-to-lot variability. To limit this variability, applicants are advised to refine the acceptance criteria of the starting material as more manufacturing experience is gained throughout clinical trial phases. Common parameters to be tested include cell number, cell viability, identity and purity of target cell population.

Traceability and labeling system

The applicant must consider establishing a labelling system that maintains the chain of identity from collection through to finished product labelling and administration. Collection bags must be labelled according to the approved policy of the collection center. A minimum of three unique identification parameters is recommended for example (donor name, DOB and donation identification number). In addition, it is important to clearly describe Batch size and batch number genealogy. 

Considerations for use of surrogate material:

It is also important to note that in many cases, applicants must use healthy donor material as surrogate for patient material for characterization and/or comparability purposes mainly due to scarcity of clinical material and patient disease states. Healthy donor materials should be segregated from patient materials in the storage area.

CAR T Cell manufacturing and testing recommendations

Manufacturing process control 

Donor-to-donor variability is a source to the cellular starting material variability. Therefore, manufacturing process should be well controlled thorough in-process control of process parameters, in-process testing and final product CQAs, as appropriate for the phase of product development.

Due to the nature of CAR T cells terminal sterilization and/or filtration is not possible. SFDA recommend applicant to manufacture CAR T cell product in aseptic environment, according to SFDA guide to good manufacturing practice for medicinal product and annex 13 manufacture of investigational medicinal product.

Analytical testing 

To ensure the safety and efficacy of CAR T cell therapies, rigorous testing is essential. This involves analyzing the product to confirm its identity, purity, potency and safety. Given the complex nature of CAR T cells, specialized assays are often needed to characterize the product. SFDA recommends that applicant start developing these assays in the early clinical trial to characterize their product. As the clinical trials progress, understanding of the products is expected to advance, therefore the identities of CQAs and their limits should be refined.

The information about assays can include:

• A description of the assay

• A brief summary of instrument calibration, maintenance and Quality control activities

• A list of assay controls

• Established and implemented written procedures

• Assay qualification and/or comprehensive validation study (before licensure) for lot release 

Analytical testing specific recommendation for CAR T: 

• Identity: It is recommended that Identity testing of CAR T cells should include assays to verify the presence of transgene (e.g., CAR expression by flow cytometry, gene detection by PCR) and an assay specific for the desired T cell markers.

• Potency: The applicant should describe and justify in the application all assays used to measure potency.  A qualified potency assay is not required to initiate early phase clinical studies; however, it must be fully validated before licensure. Consideration for potency determination of CAR T cells may account for their multiple mechanisms of action. To assess their potency, various tests, such as cell killing assays, transduction efficiency measurements, and cytokine secretion assays, can be used. Characterizing CAR T cell function throughout clinical trial phases will support comparisons and help determine the most suitable assays.

• Purity: SFDA recommended to report any unintended changes in cell numbers or types. While the rabbit test remains the standard, the LAL method is a viable option for pyrogen detection in many biological products.

• Sterility: Sterility test methods can be used per international pharmacopeia or compendial method, SFDA will generally consider the submission of other innovative testing such rapid microbiological testing, if scientifically justified. 

• Viability and Cell Number or Dose: SFDA recommended submitting data supporting a lower viability specification, demonstrating, for example, that dead cells and cell debris do not affect the safe administration of the product and/or the therapeutic effect. SFDA recommended for CART cells the applicant should have an acceptance criterion for the minimum number or percentage of genetically modified viable cells in a product lot. 

Validation studies

SFDA does not require full Process validation during clinical development. However, based on the development stage of the product, SFDA may require the applicant to provide information to support the manufacturing process consistency. Also, it is recommended to provide information to support the qualification of certain safety related or dose-related assays.

Stability studies

• Apheresis material (fresh or cryopreserved) should be assessed for stability of storage before CAR T cell manufacturing. In addition, it is recommended at least to assess cell viability of apheresis material during the storage. In case of cryopreserved apheresis material, sterility testing should be considered.

• Long-term stability, accelerated stability studies and Stress Testing Studies are recommended to determine appropriate storage periods. 

• Hold times stability: Stability studies should be performed on CAR T cells to determine appropriate holding periods. 

• In-use stability studies: in-use stability studies should be performed between final formulation and administration for fresh products or the time between thaw and administration for cryopreserved products, to support the intended hold time.

Initial stability studies can use products from healthy donors (surrogate materials).

Label recommendations

• It is recommended that you include sample or mock-up labels in Module 1 of the CTD. 

• It is recommended that the label include the product name, manufacturer information, and, as applicable, the warnings “Do not use a leukoreduction filters” and “Do not irradiate”. Labeling for licensed CAR T cells must conform to the requirements in SFDA guidelines. 

• CAR T cells manufactured from autologous starting material must be labeled “FOR AUTOLOGOUS USE ONLY”.  It is recommended that the label must state “NOT EVALUATED FOR INFECTIOUS SUBSTANCES,” 

• It is recommended that the label include at least two unique identifiers to confirm patient identification prior to administration.

• CAR T cells must also be labeled with the Biohazard legend. 

• If the results of any screening or testing performed indicate the presence of relevant communicable disease agents and/or risk factors for or clinical evidence of relevant communicable disease agents or diseases. Labeling must also bear the statement “WARNING: Reactive test results for (name of disease agent or disease),” in the case of reactive test results.

CAR T Cells Manufacturing changes

SFDA acknowledged that changes might be needed to the CAR T cell design, manufacturing process, or manufacturing facility during product development or post-approval. Changes to the final container, cytokines used during culture, or duration of cell expansion, may impact product quality, safety, efficacy, or stability. However, there are some changes (e.g., changes to the CAR construct or changing from an autologous to allogeneic product) which would generally result in a new product that requires a new Clinical trial application. 

Due to the essential role of the vector in CAR T cell activity, the impact of vector manufacturing process changes should be assessed on both the vector and the CAR T cells.

SFDA recommended to consider the following when planning on substantial changes to the vector manufacturing process and/or CAR T Cell. 

• The complexity of comparability assessments may differ depending on the extent of the change to the vector or CAR T cell manufacturing process for example: a small change in the volume of culture media to manufacture CAR T cells may generally be supported by cell viability and expansion data. In contrast, a more robust comparability study should be conducted for a change to the concentration or type of growth factors or supplements in the culture media. 

Note that the term “comparability” does not necessarily mean that pre- and post-change products are identical, but that they are highly similar and that any differences in product critical quality attributes (CQAs) have no adverse impact on CAR T cell quality, safety, or efficacy.

• CAR T cells manufactured with pre- and post-change vector should be assessed using the same cellular starting material (e.g., splitting the leukapheresis starting material from the same donor).

• When the CAR T cells or vector manufacturing facility is changed, product comparability between manufacturing facilities should be assessed.

Consideration for changes through different product development:   

Analytical comparability 

• The stage of product development may impact whether an analytical comparability study is required. during early-stage development, the major consideration should be avoiding an impact of change on product safety. However, when considering changes to be made at later stages of product development, the sponsor should evaluate the impact of the change on both safety and efficacy.

• Depending on the type of change, the applicant should consider its impact on product stability and the proposed control strategy should be submitted as an amendment to the clinical trial application before implementation of the change

Analytical comparability of CAR T cells may be assessed following the general principles described in ICH Q5E.

• SFDA emphasizes the importance of analytical comparability depending change to ensure no negative impact on CAR T cell quality, safety, or efficacy. Nonclinical or clinical studies may be requested otherwise. 

Product comparability 

• Early establishment of CQAs by characterization studies facilitates the design of comparability studies which also helps the progression of product development. 

• SFDA recommend that CAR T cell comparability be assessed using the same cellular starting material, when possible

Comparability of CAR T Cell Manufacturing facilities 

CAR T cells may be manufactured at several facilities which may contribute to product variability. In this case, you should demonstrate:

1. Comparable product is manufactured at each location to support the analysis of the clinical trial results. 

2. Analytical methods are comparable across the different sites, if applicable.

3. Each manufacturing site is following CGMPs. 

4. Using the same standard operating procedures (SOPs), personnel training program, reagents, reference materials and equipment across manufacturing facilities, when possible. 

5. IND describe any differences in the manufacturing process across the manufacturing sites.

6. Assay transfer protocol to ensure that non-compendial testing performed at each site is suitable for the intended purpose and is reproducible among all testing sites. Compendial testing methods do not require comparability studies, but feasibility should be demonstrated for each site.​