Abstract
Cell therapy has emerged as a promising new treatment in medicine, which is expected to be able to cure diseases by repairing, replacing, and regenerating tissues, as well as through immune modulation. However, challenges remain in ensuring consistent quality, clinical efficacy, and safety profiles because of the diversity of cell types and clinical indications for cell therapy products (CTPs), as well as different and complex manufacturing process. Therefore, scientific consensus and regulatory measurements are urgently warranted to promote the translation of the latest scientific advances and innovative manufacturing technologies into clinical application. This article aims to propose perspectives on the manufacturing, quality study, and quality control of CTPs and provide considerations and opinions in the regulation of CTPs.
Introduction
S
Although cell therapy products (CTP) therapies bring hopes for patients with diseases that cannot be cured by the existing pharmaceuticals, the risks of CTPs have not yet been fully understood in the aspects of cell types, heterogeneity, ex vivo manipulations, in vivo renewable, persistence and expansion, and the immune response to living cells by receptors and/or the cytokines secreted by the cells. Recently, with improved medical practice, more abundant experience, better understood mechanisms, and optimized protocols, the risks may be reduced and better controlled. Despite the variety of CTPs, the existing well-established principles of manufacturing control and quality control from the perspectives of scientific and regulatory considerations should be followed. In China, CTPs are jointly regulated by National Medical Products Administration (previously known as the China Food and Drug Administration [CFDA]), and the National Health Commission of the People's Republic of China (previously known as National Health and Family Planning Commission) based on the special properties of manufacturing, handling, and modeling of CTPs as well as the particular intrinsic characteristics of these products. Since 1999, several guidelines regarding to research and evaluation of CTPs have been published in China, and at the end of 2017, CFDA, Center for Drug Evaluation published an updated version of guideline “Guidance for Research and Evaluation of Cellular Therapy Products,” which covers the most recent technique requirements for CTPs and is regarded as a general principle and the start of series of guidelines regarding CTPs. So far, dozens of stem cell products and CAR-T products have been filed for investigational new drug approval to CFDA, and several stem cell products and three CAR-T products have already been approved for clinical trial. CTPs are at the forefront of scientific innovation, and the field is experiencing rapid technological change; therefore, the knowledge and experience of manufacturing as well as regulation are improving accordingly and should be updated and applied to practice as far as possible. Based on current knowledge, this review summarizes the updated understandings and thoughts of CTPs from a regulatory perspective in the aspects of chemistry, manufacturing, and control (CMC), including raw materials and excipients, process development and control, quality study and control, products stability, and shipment as well as environmental control measures (Table 1).
Summary of chemistry, manufacturing, and control considerations
Raw materials and excipients
Generally, raw materials refer to original cells, ancillary materials, and devices or components in the combined products. Raw materials and excipients used in CTP preparations may potentially affect the safety and quality of final products, and should be carefully selected, well defined, and thoroughly evaluated. The quality and/or grade should be suitable for the intended use. Pharmaceutical grade or regulatory authorized materials are recommended unless justified.
For the safety issue, any possibility of transmitting pathogenic viruses from raw materials and excipients should be avoided; and any residue of impurities in the final product should be limited, thoroughly evaluated, and even avoided. In some cases, toxicity study and assessment in animal models or other systems may be necessary to evaluate the safety of raw materials and excipients. High-risk materials like animal or human serum should not be used unless they are unavoidable or justified. Chemically defined media without serum and serum-free cryopreservation media have been widely used as an alternative and should be encouraged. The assessment of donor eligibility requires a thorough look at relevant medical records, physical examinations, risk factors, and clinical evidences of communicable disease agents.
Raw materials also play a key role to ensure quality consistency. Under some circumstances, the original cells and the components of culture media should be carefully evaluated, assessed, and defined to ensure the quality consistency of final products. For example, as heterogeneous cell populations with various phenotypic and functional features, MSCs are mainly dependent on individual donors, sources, and the culture conditions. Furthermore, MSCs cultured in serum from different donors display significant differences in proliferation and differentiation characteristics. 1 Therefore, the origin of MSCs and serum may be critical for this type of products and thus the use of MSC banks is recommended. Since CTPs may be genetically modified, the issues that may occur in viral vectors and transfections should be well controlled. Taking chimeric antigen receptor–modified T (CAR-T) cells as an example, quality control of viral vectors with CAR gene should be performed in order to improve purity, safety, T cell transfection efficiency, and physicochemical characterization. To acquire consistent products of CAR-T cells among individual donors, standardization of viral vectors stock is essential to produce consistent multiplicity of infection.
Since CTPs are not suitable for terminal sterilization or filtration, sterility, and endotoxin-limited assurances of excipients are critical. It is recommended that a strategy of risk assessment for the qualification of excipients should contain elements beyond what has been used in drugs and other biological products, including that each component of excipients should be sterilized and tested for endotoxin level to standardize appropriate acceptance limits. Furthermore, it is possible that the excipients could interact with cells or media components and that the cell functions may thus be affected. Therefore, evaluations or tests for these interactions are necessary. Some excipients in CTPs have already been approved by FDA, such as sodium chloride, dimethyl sulfoxide (DMSO), dextran 40, and bovine type I collagen. 2 For these established excipients in the CTPs, formulations are required to evaluate the potential toxicity when a larger amount or a new administration route is applied. In some cases, the bridging safety and toxicological studies should also be conducted. For example, DMSO, which is frequently used in cryopreservation, has been demonstrated to have toxic effects that may lead to adverse outcomes in patients, such as nausea, vomiting, or even death. 3 Therefore, careful selection of DMSO in final products and/or the handling of DMSO products before administration should be noted. For the excipients that have never been used in CTP, the scientific justifications as well as the toxicity studies are required to demonstrate its safety on living cells.
In some CTPs, certain devices or components of combination products will come into direct contact with cells and be part of the final products. In such cases, the quality of device and the compatibility with cells should be fully evaluated or tested.
Process development and control
The development of robust and well-characterized processes for products is critical. Different from the classic biological molecules which usually have a general process to ensure sufficient quality and potency for early clinical trials, CTPs are heterogeneity in technology and model (such as decentralized or centralized). Therefore, a reliable, robust, and economical process for CTPs will lead to satisfactory consistencies between doses and individuals and will ensure successful development. Collectively, the scalability, automation, intermediate product stability, sterilization operation room, process control, and general process robustness/failure rate and supply chain could all affect the quality of the final product and should be taken into consideration.
Identifying and refining manufacturing system
Manufacturing process of CTPs refers to the process starting from isolation of original donor cells and ending with administration of processed cells into patients. The manufacturing process mainly consists of cell isolation, cell characterization, cell induction or gene modification, cell culture or amplification, purification, testing, storage, and delivery.
Before designing the manufacturing process, the precise definition of clinical indication should be clarified, and the specific cells with the required functions or mechanism should be formulated. 4 The parameters of each procedure in the manufacturing process could be adjusted during the development process according to the most updated information in response to new knowledge, such as the scale-up of manufacturing, optimization of culture media, and removal of impurities. Furthermore, the critical parameters in the process should be determined and defined. For example, culture conditions such as temperature, pH, and dissolved oxygen are key parameters that may affect cell growth kinetics, cell age, and cytokines secreted by cells. It is reported that the agitation keeping homogeneous culture conditions may change the phenotype of shear-sensitive cells. Dissolved oxygen may also has an effect on both the potency and purity of pluripotent stem cell (PSCs), hematopoietic stem/progenitor cells, and MSCs, 5 as researches have shown that the level of oxygen and the concentration of fibroblast growth factor 2 in the culture media will determine the differentiation capacity and phenotype of MSCs. 4 Even the accumulation of metabolic byproducts, such as lactate or ammonium, as well as decreased pH may be associated to the inhibition of cell growth and the loss of PSC phenotype. 6 Media exchange rate and different culture strategy (fed-batch or perfusion) may affect the accumulation of waste products and factors secreted. Additionally, the cooling rate of cryopreserved cells is vital to cell survival rate. A strict management of aseptic and automatic operations of bioreactors and/or purification is essential for manufacturing as well, especially for the large-scale manufacturing. As for CAR-T cells, the transfection efficiency of CAR, parameters such as the acceptable ranges of gene-modified CAR-T ratios among total T cells, and quantification of copy numbers of the CAR gene per cell should be taken into consideration and well controlled. Above all, proper identification and refinement of the manufacturing system is essential to target the production of desired and qualified products. Some researchers have brought up the concept of “quality by design” to design and develop the process, yet it has not been fully applied to the field of CTP due to the inherent variability and undefined maturity of biological systems. However, as products and processes are becoming increasingly well established, the concept will become useful to drive efficiencies. 1,7 Besides, the extend and complexity of ex vivo operations, results, and trends of laboratory controls as well as feedback from clinical trials are critically important for the successful development of CTPs and should be taken into account when designing and streamlining the manufacturing process.
Manufacturing environment
The environment of the manufacturing process needs to meet the related requirements, which are necessary for regulatory assessment and consistency among batches or individuals. Good manufacturing process (GMP) is usually recommended as a mandatory and risk-based approach according to different product types and procedure complexity should be taken into consideration. For CTPs, the whole manufacturing process should be conducted aseptically and according to different manufacturing systems (closed or open system), different grade of clean area will be required. For the manufacturing of viral vectors, proper control measures should be implemented to avoid the risk of contamination. The viral or infected materials in the conditions involving the infected donors and replication competent vectors should be prevented to spread and be kept in tight control. Furthermore, adequate measures should be taken to ensure products traceability and avoid cross-contamination and mix-ups.
For the manufacturing facilities, the automation techniques and in-line sensor systems will be expected to help improve quality control and minimize the run-to-run variability, especially for large-scale manufacturing of allogeneic therapies.
Manufacturing scale
The manufacturing scale to fulfill product demand should be considered. In order to meet the clinical need, the amount and frequency of production will be determined by the dose of cells per patient, cell characteristics, product stability, and life cycle of products. “Scale-out” for an autologous product requires the ability to carry out a small size process for many times, whereas “scale-up” for an unmatched allogeneic process requires the ability to carry out a large size process only for a few times. The approaches associated with the scale-up manufacturing include increasing bioreactor size, transforming static processes to suspension cultures, and optimization of solid attachment substances (e.g., microcarriers or other structure) for adherent cells. The key challenges for scaling up cell cultures are to maintain key quality attributes of cells such as identity, potency, purity, safety, metabolism, and cell behavior, or clinical performances such as therapeutic potency and treatment efficacy. The limit of doubling time during culture process varies according to cell types, so it should be ensured that the cells retain function or potency without senescence, slowing of growth rates, differentiation, or loss of phenotype. In addition, maintaining homogenous cell suspension and relatively short process time during final formulation and filling are essential in the manufacturing scale design and evaluation process.
Robust process and changes
Robust manufacturing process should be developed to ensure the product quality to be adherent with the predesigned acceptable level, such as an appropriate limit, range, or distribution. Thus, properties of raw materials should be well defined and critical manufacturing parameters should be controlled. For example, homogeneity in parameters of the culture system is crucial to the cell growth, including fresh nutrients, oxygen, signals from growth factors and/or serum, and removal of waste. To ensure the consistency of allogeneic CTPs, establishment of cell banks kept as seeds for repeated bathes to enable bath-to-bath consistency is recommended. Equally, donor-to-donor variability in individual therapies and run-to-run variability in allogeneic therapies may be handled with proper investigation and control on the systematic manufacturing process to minimize the variability.
Once any parameter in the manufacturing process changes, product comparability study is necessary to demonstrate that the safety, identity, purity, or potency of the products are not affected. Comparability study should be based on risk mitigation strategy, and changes with high risks would require more data including experimental data clinical data to support the rationality of changes proposed. It is recommended to define the critical quality attributes for evaluation, same as the case in traditional biological pharmaceuticals. For example, changes of the chemically defined media or dosage reduction of media components should be thoroughly validated and evaluated in terms of cell attributes and residues profile. For the substantial changes, it is required to file the changes and comparability study data to regulatory authorities before any actions are carried out or it is recommended to communicate with the regulatory authorities for comments and advise.
Quality study and control
The diversity of CTPs and of the materials used makes it difficult to recommend specific protocols for the CTP qualification program which should be developed case by case for each product. Generally, product quality qualification program should include product quality study and product quality control. Besides, the quality of manufacturing facilities, process, validation, and raw materials should comply with GMP requirements and relevant regulations.
Quality study
Quality study refers to comprehensive research and analysis of the quality profile of CTPs which generally include safety, potency, identity, and purity etc. Quality study is usually conducted before and during clinical trials as well as when any changes which could potentially impact product quality may occur, like changes of raw materials, process parameters and manufacturing site, etc. Usually, not only the final products but also the bulk products or the intermediates could be included into the study. The data obtained by monitoring, collecting, and analyzing in the quality study could be used to demonstrate the safety and efficacy profile of a product, and could also be used to prove the quality design of a product. Besides, the acceptance criteria of product release specification could be justified via quality study. The study actions and data above should be adequately filed in the product documents, and the trend analysis is necessary at proper intervals. Moreover, the batches selected for quality study should be representative of those that would be used in the clinical trial.
Product safety: The safety issues related to CTPs include contamination, cell malignant transformation, off-target toxicities, and tumorigenicity and tumor growth promotion, as well as recovery mutation of viral vectors if they are applied. Contamination may originate from microorganisms (bacterial, fungal, and mycoplasmal), endotoxins, cell line cross-contamination, non-cell particulates (like plastic fragments, residual microcarriers, and fibers), and ancillary material residues. The contaminations should be controlled in the raw materials, the manufacturing process, and the final product residual assay, in which the acceptable limit should be defined. Unexpected cell changes, like malignant transformation, may present as beyond the limit of cell passage number. Therefore, cell culture time span and cell passage limit should be well studied and controlled. Off-target toxicities and unexpected T cell functions in vivo are the challenges for CAR-T products. In order to identify tumor-specific surface antigens and achieve the expected effects, some innovative designs of CARs have been invented, such as the inhibitory CARs (iCARs), switchable CARs (sCARs), and multichain CARs (mcCARs), with the function of introducing suicide genes for CAR gene transfer and/or surface co-expression of binding epitopes for depleting antibodies in the CAR-product to mitigate unexpected off-target toxicities, and/or T cell trafficking and/or persistence. 8 Therefore, correct expression profile of genes on target cells is very important for the gene transfer cell therapy product and should be thoroughly identified and/or quantified properly in final products.
Identity: Cell identity is more complex than proteins or small-molecule drugs and is typically demonstrated through the presence or absence of cell surface markers, which are expected to correlate with functional activity, and sometimes karyotype is tested. In some cases, several different cell identity tests may be conducted for a single product, for example, the combined MSCs have varying properties from different tissue sources so more than one cell identity test may be required. 9 CAR-T products are necessary to demonstrate chimeric antigen receptor and T-cell marker double-positive cells in order to discriminate against untransduced T cells and undesirable cell types.
Potency: A potency test should be carried out according to the mechanism of action or effect. For example, the potency test of MSC could be the assays describing the engraftment and tissue formation, or the secretion of paracrine factors. Potency of CAR-T cells would be measured by the ability to destroy the target cells and cytokine release, proliferation, and/or degranulation.
Strength: Traditionally, strength refers to the quantity of active ingredients in a dose of treatment. For CTPs, strength could be cell subpopulation, density, number, and/or viability. Strength could be measured in different assays, and manufacturers/licence holders/sponsors should demonstrate the most appropriate kind of assay. Other factors, such as population composition, mitochondrial content or activity, substrate abundance, and redox state of growth medium should be properly taken into consideration as well. 5
Purity: Impurities in CTPs can present as undesirable cells, contamination, ancillary materials residues and particulates etc. It is reported that impurities of CTPs may cause compromised effects and immune complications. Therefore, purity thresholds should be set up and confirmed by testing results from animals and/or humans. Thresholds for purity may vary according to the products. It has been reported that as few as 1/4000 residual undifferentiated PSCs can lead to the formation of teratoma, which is beyond the scope of detection by flow cytometry–based phenotypic assays. 10 In CAR-T cell therapies, however, efficacy may be affected by the proportions of proliferative naive or central-memory T cells in the final product. Thereby, in the purity analysis, unwanted cell types and off-target effects of cells should be quantitatively and qualitatively analyzed and should be kept in tight control if necessary.
Quality control
In addition to quality study, quality control should be applied to each lot. Quality control is intended used to ensure relevant tests are carried out and then the quality is judged satisfactory to release for use, sale, or supply. Quality control should be conducted not only at the end of production, but also throughout the whole manufacturing process. It is not sufficient to assure quality just by testing on final products via the measurable release criteria; instead, it has to be accomplished in a step-by-step manner or monitoring by in-process control at appropriate stages of production to control those conditions that are critical for the quality and safety of the product. The qualified person should ensure that the quality control is carried out under suitable conditions and procedures in accordance with GMP and with proper testing methods that have been justified and validated. Precautions should be paid to avoid any risk for the products when in-process control that may carried out within the production area.
In a more general way, quality study and quality control may not only refer to the finished products, but also to the raw materials, intermediate product, and packing materials, etc. Manufacturer/License holder/Sponsors should establish the strategies of quality control or the mitigation measures having regard to the above which will ensure the quality, safety, and consistency of the final products. The extent of control increases as clinical development progresses and it is recommended that the full quality control should be well established and be complied by the time phase 3 clinical trials begin.
The test methods used to ensure the final products/intermediates meet release acceptance criteria are designed and adapted to meet the specific characteristics of CTPs. Test methods are recommended to be developed based on the best available science and should be robust, reliable, and capable of being validated and should provide results before release for clinical use or alternative methods could be applied unless justified.
Stability, packaging, and shipment
Developing reliable storage and transporting method with cell efficacy at a reasonable and economical cost for CTPs is a challenge. Once the cells in the final product have been harvested, they are kept away from their ideal environment without nutrition or suitable conditions and will lose viability or activity over a certain time. Therefore, the development success of CTPs is potentially affected by limitations in stability.
Generally, CTPs may be maintained in liquid suspensions until administration or be cryopreserved, which is the suitable approach depending on the products stability, autologous or allogeneic use, and/or manufacturing model. Fresh shipment of liquid suspensions is usually possessed with short shelf-life and is not considered suitable for large-scale manufacturing. For the cryopreserved products, factors such as phase transition, osmotic intolerance, cryoprotectants, and warming/thawing temperature could cause cell injury, 11 and should be evaluated regarding cell survival and functioning in order to develop an optimized cryopreservation protocol for efficient manufacturing and banking of cells in different stages. Poor cryopreservation protocols will result in a significant post-thaw cell loss and limit the total dose available for administration. Furthermore, given the fact that the cells are from various tissues and various donors and may react differently to cryopreservation, careful design of the cryopreservation protocols for particular cell types is recommended. Besides, the type and concentration of cryoprotectants and the cryopreservation container used for storage should also be taken into consideration. For example, DMSO is the most commonly used cryopreservation product. However, DMSO is potentially cytotoxic and can have adverse systemic effects in vivo when ingested. The direct use of frozen-thawed cells with DMSO in clinical practice is reported to have caused mild to serious adverse reactions. 3 Therefore, the removal of DMSO from the final product in the clinical setting is considered if large volumes of DMSO are included (50 mg per day or less is acceptable according to ICH Q3C Impurities: guideline for residual solvents [Q3C] or case by case). The washing process prior to administration of cryopreserved cells is put in place to ensure adequate recovery of cell functions and removal of cryopreservation components. In contrast, some products may be administered to patients in the freezing media to minimize manipulations of the cryopreserved cells, such as mechanical forces, osmotic stress, cell packing/clumping, and potential cell loss and due to the difficulty of creating a sterilized condition in the clinical setting. Therefore, further evaluations on the necessity to remove cryoprotectants are warranted. Once the preservation conditions are established, it is required that at least one batch of the product, unless justified, is included in the stability program per year for trend analysis.
Additionally, the product storage containers and transport logistics process can vary significantly and eventually affect product quality. CTPs should be suitably packaged to maintain product quality and ensure product integrity. The storage container, especially the primary one that will directly contact the product, should be carefully evaluated to ensure that it has the capacity to withstand outside temperatures and fulfill the light-sealed and liquid-sealed requirements, and product characteristics, storage, handling, and shipping conditions as well as maintenance of suppliers should also be taken into consideration. They should also be compatible with the freezing solvent and prevent cell attachment to the tubing or ports of the containers. For example, when DMSO is applied as a cryoprotectant, the compatibility of DMSO with container materials should be carefully evaluated, because DMSO has been reported to interact with many polymeric materials, which may result in an incompatibility with the packing materials. 12 Furthermore, the products should be properly labeled on the containers or on the packaging and the labels should be well designed and easy to identify as well as compatible with transport and storage conditions. When labeling, special precautions should be taken for the investigational products to avoid a mix-up where blinded products are used or different products are handled on the same packaging line at the same time.
The shipment for CTPs should be regarded of same importance as the manufacturing process, because shipment will impact the product quality as the end of the chain. Therefore, it is recommended to validate the shipment process, including temperature, humidity, transportation, route, secondary packaging, duration, and monitoring system.
Environmental control measures
CTPs containing genetically modified organisms may require additional control measures considering the risk that may pose for the environment. The design and establishment of the premises, organization and facilities in this case should control the risk tightly and handle the residues of the production properly. The containment measures and the emergency plans should be taken into account. Take CAR-T, for example: where replication-limited viral vectors are used, risk assessment and mitigation measures should implemented to prevent formation of replication competent recombinant vectors.
Discussion
CTPs are regulated as drugs rather than clinical practices in most countries and areas, while the distinguishing characteristics and the still-unknown risks of CTPs—which are unique and different from traditional biological products—require more flexible regulatory requirements and comprehensive risk management. Raw materials used in CTPs production should be strictly selected, evaluated, and controlled; contamination that could introduced into the production system should be avoided. The manufacturing process should be well designed, well refined, well validated, and be conducted aseptically in accordance with GMP with the proper grade of clean areas. Quality systems should be established to ensure that the quality of products can be adequately controlled with proper acceptance criteria during the process and for product release specifications. The testing methods used to monitor product quality should be developed according to the specific properties of products, and alternative methods may be applied other than the pharmacopeia ones when justified. Moreover, the stability testing should be carried out to demonstrate that the storage and shipment conditions are suitable and that the product is compatible with the packaging materials. Throughout the lifecycle of CTPs, manufacturing activities may change as products move from clinical trials to licensure and commercialization as well as after commercialization. In the early stages of development, safety concerns should be the primary focus, and in the later stages, manufacturing activities should be completely developed and should comply with the regulatory requirements. Meanwhile, the quality comparability bridging study should be conducted to demonstrate the consistent quality profile of the products produced before and after the changes.
The clinical success of CTPs in the recent years, especially the use of CD19-positive CAR-T cells in the treatment of acute lymphocytic leukemia and non-Hodgkin lymphoma have brought a big boost in the field of immunotherapy and led to significant capital funding in the biotech sector, as well as promoting new discoveries in basic research that may be put into translational use and commercial development. Such rapid development provides not only new opportunities for unmet clinical need, but also challenges for researchers and regulators with limited knowledge and experience. Given the complexity of this type of product and the challenge of consecutive application of various new techniques in this field, a case-by-case risk-and-benefit evaluation principle is recommended. In addition, sponsors are encouraged to closely communicate with regulators on the above scientific issues. Regulatory authorities should define regulations and requirements for clinical application of CTPs; meanwhile, flexible evaluation may be applied considering the intrinsic heterogeneity property of these products, so that patients who are seriously ill without efficient drugs may have the chance to be properly treated. In China, there are no CTPs commercially available so far; however, there are several promising and world leading products that are in the stage of clinical trial, and the primary clinical data are very optimistic.
Despite the ongoing controversies and challenges, it is becoming increasingly clear that with further improvement in product design, manufacturing, and testing techniques, as well as a better understanding of cell culture and cell population regulation, the future of cell therapy is bright and promising. Besides, the cost will be decreased, while quality is maintained or even improved. Researchers and clinicians should collaborate to promote the translation from bench to bedside. All of these actions will contribute to better investigation and utilization of CTPs in the future.
Footnotes
Acknowledgments
We thank Professor Wan Tao and Bai Yu for very helpful suggestions and comments on the manuscript.
Author Disclosure
No competing financial interests exist.