Regulatory considerations for developing a phase I investigational new drug application for autologous induced pluripotent stem cells‐based therapy product

Abstract Induced pluripotent stem cells (iPSC)‐based therapies have been hailed as the future of regenerative medicine because of their potential to provide treatment options for most degenerative diseases. A key promise of iPSC‐based therapies is the possibility of an autologous transplant that may engraft better in the longer‐term due to its compatibility with the patient's immune system. Despite over a decade of research, clinical translation of autologous iPSC‐based therapies has been slow—partly due to a lacking pre‐defined regulatory path. Here, we outline regulatory considerations for developing an autologous iPSC‐based product and challenges associated with the clinical manufacturing of autologous iPSCs and their derivatives. These challenges include donor tissue source, reprogramming methods, heterogeneity of differentiated cells, controls for the manufacturing process, and preclinical considerations. A robust manufacturing process with appropriate quality controls and well‐informed, prospectively designed preclinical studies provide a path toward successful approval of autologous iPSC‐based therapies.

Autologous cell therapy requires a new round of product manufacturing for each patient, which increases logistical challenges and costs associated with the manufacturing process. 18,19 But an advantage of autologous cell therapy is that the product engraftment in patients may not require the use of long-term systemic immunosuppression as compared to an allogeneic cell therapy product that relies on the immunosuppression of patients to achieve longer-term engraftment. [20][21][22] Long-term systemic immunosuppression is associated with serious adverse events, like an increased risk of infections or cardiovascular disorders-especially in older patients. 23 Furthermore, immunosuppression discontinuation that may inadvertently happen in some patients will likely compromise graft survival. 22,24 This one key difference may significantly improve clinical outcomes of autologous cell therapy products as compared to allogeneic products and have favored the continued use of autologous products, despite their seemingly high cost. The approach for developing autologous and allogeneic iPSC-based therapies is fundamentally different in several aspects, including the proof of concept, manufacturing workflow, preclinical study planning, regulatory approach, and the clinical strategy. 23,24 All of these differences affect the overall design of investigational new drug (IND)-enabling studies, clinical trial design, market approval, financial feasibility, and commercialization strategies for autologous cell therapy products.

In the United States, the Center for Biologics Evaluation and
Research at the FDA is responsible for regulating cell-based therapies. 25 The FDA has issued guidelines in the Code of For a checklist of the information required by the U.S. FDA to compile a phase I IND application, see Table 1, including required sections in (a) chemistry, manufacturing and controls, (b) non-clinical information, and (c) clinical study plan. For details of an approval path for cell therapy BLA, see Creasey et al. 26 To date, none of the ESC-or iPSC-based therapies have reached the stage of BLA submission for market approval. Presently, there are four FDA-approved clinical trials in phase I/II in the U.S. testing iPSCderived products. 27 The therapeutic effect of these iPSC-based therapies is being evaluated for different diseases, including age-related macular degeneration, advanced solid tumors including lymphoma, relapsed/refractory acute myeloid leukemia and B-cell lymphoma, and chronic heart failure. Internationally, there are six additional iPSCderived products in clinical studies-in Japan, China, and Australia, for details see Martín-Ibáñez and Sareen. 27 With the continued development of autologous iPSC-based therapies, there is a need to develop a regulatory roadmap for manufacturing and preclinical studies required to complete a phase I IND-application. The challenges in the development process of autologous iPSC-based products include establishing tissue and donor source, the heterogeneous phenotype of cells, elaborate manufacturing process, intricate in-process quality controls, cryopreservation of intermediate and/or the final product, need for detailed product characterization, and short shelf life of a live product. 20,23 These challenges can markedly influence the cell therapy product profile and need to be adequately addressed in the early stages of product

Significance statement
The induced pluripotent stem cells (iPSC) field has developed remarkably in the last decade, with some cell-based therapies already in the clinic. However, there are still many hurdles to overcome before iPSCs attain their full clinical potential. Despite manufacturing challenges, autologous iPSC-based cell therapies are being tested for various diseases. Clinical data from autologous stem cell therapies have suggested limited immune rejection and reduced necessity for postoperative immunosuppression. Autologous cellbased therapies have their own set of regulatory requirements that need to be acknowledged and addressed to translate these products successfully to the clinic. A better understanding of an autologous stem cell therapy product and development of a robust manufacturing pipeline with safe and efficacious preclinical endpoints will help us develop reliable approaches to get autologous cell therapies commercially approved for unmet clinical needs in the near future.
development. Additional manufacturing process controls involve validation of analytical testing methods employed for the in-process and final product release assays, including assay specificity, sensitivity, accuracy, and reproducibility; this is essential to ensure batch-tobatch consistency and product comparability between multiple runs. 17,24 Establishing robust and standard practices around manufacturing, handling, delivery, shipping, and storage of the cell therapy product will help ease the path to market approval and also reduce manufacturing expenses in the long run. The main factor for the potentially higher cost of autologous cell therapy products as compared to allogeneic cell therapy products is the need to repeat the manufacturing process from start-to-finish for every patient.
Manufacturing process repetition increases labor, facility operation, and consumable cost. 28 Several of these costs can be lessened and controlled by the use of automation. Automated bioreactors and cell culturing robots combined with artificial intelligence-based product analysis tools are being adapted for iPSC-based therapies. [29][30][31] Because of the smaller scale of manufacturing, autologous cell therapies are particularly amenable to scaled out automation. It is worth noting that since currently there is no commercially approved autologous or allogeneic iPSC-derived cell therapy product, manufacturing cost can only be conceptualized.

| Starting material
One of the first quality checks to be put in place for an autologous iPSC-manufacturing pipeline is the starting material. In the case of an iPSC-derived product, it is donor cells derived from blood, skin fibroblasts, or any other somatic cell type. Centers for Disease Control and Prevention (CDC) and the American Association of Blood Banks (AABB) recommend a list of specific disease pathogens for which any donor, including a patient, should be screened for before blood collection. 33,34 This guideline can help with autologous iPSC-product manufacturing as well. Donors that test positive for any of the pathogens listed below may be excluded from the study. This exclusion ensures that these pathogens do not propagate from donor material into the manufacturing workflow and to other cell therapy products.
The pathogens and tests for their detection listed by CDC and AABB are:

| Cell source
iPSCs can be generated by reprogramming of any somatic cell. 35 But for generating a cell therapy product, the starting cell source may be relevant. The ongoing clinical trials have mainly used skin fibroblasts and peripheral blood CD34+ cells, for ease of cell isolation, iPSC manufacturing, and the quality of derived iPSCs. 32,36 As of now, there is no regulatory guidance available for the choice of a given somatic cell type. CD34+ cells have been demonstrated to have a higher reprogramming efficiency as compared to terminally differentiated blood cells, likely because these cells are already in a stem cell state, and their chromatin is better poised to reprogram into a fully pluripotent state. 37 This cell type has resulted in the development of a highly reproducible autologous iPSC-manufacturing process. 32 Although there is a relatively lower yield of CD34+ cells from peripheral blood as compared to the cord blood, peripheral blood is easily obtainable from any patient and provides one of the least invasive cell sources for autologous iPSC generation. 32,38 Moreover, GMP-compliant protocols have been developed to expand CD34+ cells to a sufficient number required for the iPSC reprogramming process. 32,39 In conclusion, the choice of starting cell source is flexible for an autologous cell therapy product with certain advantages provided by CD34+ cells.

| iPSC reprogramming technique
An essential requirement for the iPSC reprogramming technique used in a clinical manufacturing process is the reproducible and efficient generation of fully-pluripotent iPSCs with zero genomic "footprint" (no leftover traces of reprogramming factors in the host genome). First-ever reprogramming into iPSCs was performed using four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC, traditionally called the Yamanaka factors. [11][12][13] These transcription factors were delivered using a retroviral system, a method that leads to the integration of reprogramming factors into the transduced cell's genome. 40 Such a reprogramming system, if used in generating a cell therapy product, will significantly increase scrutiny for regulatory approval.
However, the reprogramming field has been evolving fast, and presently several zero genomic footprint reprogramming methods are available, including episomal plasmids, Sendai virus, adenovirus, minicircles, and miRNA, mRNA or protein-based overexpression of reprogramming factors. [41][42][43][44][45][46][47][48][49] There is limited data on the cost and validation of these zero-footprint reprogramming techniques, especially when used for clinical-grade manufacturing, but they all seem to work well to generate iPSCs. 40 Independent of the reprogramming method used, a critical requirement for this step is to demonstrate the loss of these reprogramming substrates (zero footprints) because the continued presence of such factors may increase the tumorigenic potential of the final product. athy. 50 If a USP or GMP-compliant AM is not available-AM may still be used in the human phase I trial, but such AMs need to be switched to at least a GMP-compliant version before phase II. 52 AMs are one of the essential components of an iPSC manufacturing process. The choice of a correct category of reagent is critical for accelerated regulatory approval.

| Cryopreservation and intermediate stocks
Autologous iPSC-derived product manufacturing can often extend from weeks to months. 8

| Product characterization and in-process controls
Critical Quality Attributes (CQAs) are measurable properties of a cell therapy product that help better characterize the product. CQAs are especially helpful for autologous products because they help understand variability and its source within the manufacturing process, determine the allowable limit of variability from batch-to-batch, and control this variability. These attributes are also referred to as inprocess QC checks (tests performed in the intermediates stages of product manufacturing) and release tests (tests performed on the DP prior to its release for patient administration). reprogramming. This assay can be performed using any commercially available qPCR-based assays. The most critical aspect of this assay is to determine its lower limit of detection and ensure that the assay is sensitive enough to detect less than one copy of the reprogramming system per cell.

Genomic stability:
Previous work has suggested that during the reprogramming and/or cell passaging process, iPSCs may become genomically unstable, acquire karyotypic abnormalities, and/or may copy-number variations and mutations. 36,61,62 All of these changes may cause the final product to become tumorigenic or acquire an unstable or incomplete phenotype. iPSC karyotyping can be checked using the Gband karyotyping assay, and oncogenic mutation discovery is possible using targeted sequencing of cancer-related genes commonly mutated or rearranged in human cancers. 63  6.1 Sterility, Endotoxin, and Product Identity Tests: These attributes were discussed in detail in points 1, 2, and 3 above.
6.2 Purity: Cellular composition of the DP, including non-desired cell types, especially pluripotent cells, is critical to be determined to confirm the purity of the product. The iPSC presence can be easily detected using flow or qPCR-based assays. 66 However, the presence of non-desired non-iPSCs is hard to determine as their lineage is also unknown. Assays like scRNAseq may be used to address this specific problem. 67 It is well known that pure iPSCs are prone to teratoma formation, and a low number of iPSCs can form a teratoma. [68][69][70] Because of this, it is important to determine the lower limit of detection of iPSC detecting assays. alter the disease course. The potency of a product is related to its measured efficacy in vitro and in vivo in animal models.
Efficacy assays will vary from product to product, and various techniques, including artificial intelligence, may be used to determine these potency assays. 31,32 Independent of the kind of assay, it is critical that the potency assay used is validated before the product reaches the phase II clinical trial.

| PRECLINICAL STUDIES: PRODUCT SAFETY AND EFFICACY
For any IND-application to be activated to test a product in patients, there is a regulatory requirement to confirm the safety, and if possible, its efficacy. These data are collected in preclinical studies, which preferably should be Good Laboratory Practice (GLP)-compliant (details available in 21 CFR Part 58 of the FDA and in Table 3). If these studies cannot be GLP-compliant, a justification for non-compliance may be required. Preclinical studies include in vitro and in vivo data. 72 in vitro studies are performed to qualify manufacturing process reproducibility, product purity and safety, functional characteristics, and

T A B L E 3 Pre-clinical study requirements for a phase I IND application
General study information Test system and study design Results  32 Here, we provide an overview of some of the standard preclinical studies. Due to the product-specific nature of efficacy studies, those will not be discussed in much detail here.

| Reproducibility of the manufacturing process and product characteristics
For autologous cell therapy, the process is the product, that is, confirming the manufacturing process reproducibility is a crucial part of the preclinical studies. 73,74 Once the research-grade process is translated into a GMP-facility, successful cGMP-compliant manufacturing of the proposed clinical product needs to be demonstrated in the IND application, preferably from multiple patients. This exercise serves multiple purposes: it helps set release criteria that are widely applicable for the product derived from multiple patients; it helps train operators on the cGMP-compliant manufacturing process; it helps better understand the range in which product CQAs fall when the product is manufactured from different patients; it helps define SOPs for a cGMP-complaint process; and it helps manufacture sufficient product for preclinical studies.

| Removal of impurities
Often, cell therapy products are cultured in media that contain recombinant proteins, chemicals, buffers, and serum (or cryo-protectant if delivered frozen to the surgery suite). Such impurities can cause inflammation or toxicity systemically or at the site of transplantation.
Therefore, the removal of such impurities may be required before transplantation. 71 This can be easily achieved by several sequential washing steps. Removal can be demonstrated by calculation of the amount of a given impurity after subsequent dilutions and/or by specific assay like mass spectrometry.

| In vitro safety
Leftover iPSCs in the DP is a major concern of regulatory authorities.
Besides demonstrating the absence of iPSCs in the DP, in-vitro "spiking" studies can be performed to demonstrate the non-survival of iPSCs in the differentiation process. This assay is based on the hypothesis that iPSCs require special culture medium and cannot grow in a culture medium that includes targeting product-specific differentiation factors. Following test groups can be used in the assay (a) 100% target cells, (b) 100% iPSCs (positive control), (c) 99% target cells mixed with 1% iPSCs, and (d) 90% target cells mixed with 10% iPSCs. Cells in these four groups are cultured using target product differentiating conditions. 32 Techniques like flow cytometry, qPCR, and scRNAseq may be performed to determine surviving iPSC or sporadically formed cells of a different lineage. This assay provides additional confidence in the safety of the cell therapy product.

| In-vitro stability of the clinical product
After washing the cell culture medium, an excipient is added to the cell therapy product to act as a preservative prior to the release of the DP from the GMP-compliant manufacturing suite. This excipient is to be used to store, transport, and administer the product. Thus, the choice of the excipient is very crucial to ensure that it is compatible with the cells and, importantly, is permitted or approved by the FDA to be administered in humans. One of the options to use as an excipient is isotonic saline. However, live cell therapy products may have a relatively short shelf life in the excipient used for dose administration and transport. Thus, it is crucial to determine the duration for which the clinical product is stable with optimal cell viability in the excipient, and in the delivery device. Product stability should be determined in its transportation container system and when loaded inside the transplantation device (Table 3). 71 This study gives surgeons confidence about the product's shelf life while they prepare the patient for the surgery.

| Preclinical toxicity and biodistribution
One of the main concerns for any new cell therapy product is its safety profile -this includes non-teratogenic/tumorigenic potential, any local or systemic toxicity, and non-targeted migration of the transplanted cells. 21,75-78 A phase I IND-application of a stem cellderived product may not be approved without sufficient data on these three characteristics of the product. Different animal models can be used in preclinical studies to ensure that the transplanted human cells (xenograft) survive long enough to reveal their tumorigenic potential. 79 It is the sponsor's responsibility to justify the suitability of chosen animal models based on the test article route, site of administration, its dosage, and long-term survival. Preclinical studies need to be conducted using the dosage and delivery route that is representative of the regimen to be used in patients. The product in preclinical studies should be manufactured using the same manufacturing process, which will be used for product manufacturing during the clinical trial to demonstrate that the product proposed to be transplanted in humans has been thoroughly investigated in in-vitro studies and animal models.
Furthermore, for an autologous iPSC-derived product, cells derived from multiple (2 or more) donors may need to be tested in animals. One of the most critical requirements for such preclinical studies is that they should be GLP-compliant with prospective study plans. Refer to Table 3 for an outline of the GLP-compliant preclinical study design for an autologous iPSC-derived cell-therapy product.
It is worth noting that although preclinical animal testing can de-risk an iPSC-based cell therapy product to some extent, it cannot ascertain that the safety profile of human cells obtained from transplantation performed in animals will actually translate to patients. Therefore, prospective risk-assessment and riskmanagement of cell therapy products are quint-essential. This may be done by a justification of dosage, delivery site, delivery route, disease stage, combined with data about the purity of the DP. If the DP is composed of post-mitotic cells of only one lineage and is relatively free of non-desired cells, safety risk associated with cells lessens significantly. But for products that contain a mixed population or stage of cells, the progenitor stage may contain pluripotent or multipotent cells. Teratoma formation has been detected with as low as 245 pluripotent cells. 80 Thus, for products with a mixed population or stage of cells, a prospective risk-management may be required in the clinical protocol, despite a demonstrated safety profile in preclinical studies.

| Clinical considerations
A detailed discussion on clinical considerations for cell-based products is beyond the scope of this article because of the uniqueness of clinical aspects of different disease indications. Patient safety is of paramount importance, and, in part, it is ensured by an institutional review board (IRB) and data safety monitoring board, in addition to the FDA approval of the IND-application. To maintain the legitimacy of the trial, patients must not be incentivized or coaxed into the trial; rather, they should be enrolled using an IRB-approved informed consent form. Because a phase I study by design is a safety trial, the first patient cohort should be chosen such that if the drug product fails its safety profile, it causes minimal or no harm to patients. Patients must be clearly informed of the potential risk associated with the first-inhuman procedure. 81

CONCLUSION
The iPSC field has developed remarkably in the last decade, with some cell-based therapies already in the clinic. However, there are still many hurdles to overcome before iPSCs attain their full clinical potential.
Despite manufacturing challenges, autologous iPSC-based cell therapies are being tested for various diseases. Clinical data from autologous stem cell therapies have suggested limited immune rejection and reduced necessity for postoperative immunosuppression. Autologous cell-based therapies have their own set of regulatory requirements that need to be acknowledged and addressed to translate these products successfully to the clinic. A better understanding of an autologous stem cell therapy product and development of a robust manufacturing pipeline with safe and efficacious preclinical endpoints will help us develop reliable approaches to get autologous cell therapies commercially approved for unmet clinical needs in the near future.