Bioprocessing considerations for generation of iPSCs intended for clinical application: perspectives from the ISCT Emerging Regenerative Medicine Technology working group.

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes.

Mitigating Deficiencies in Evidence during Regulatory Assessments of Advanced Therapies: A Comparative Study with Other Biologicals.

Advanced therapy medicinal products (ATMPs) comprising cell therapy, gene therapy, and tissue-engineered products, offer a multitude of novel therapeutic approaches to a wide range of severe and debilitating diseases. To date, several advanced therapies have received marketing authorization for a variety of indications. However, some products showed disappointing market performance, leading to their withdrawal. The available evidence for quality, safety, and efficacy at product launch can play a crucial rule in their market success. To evaluate the sufficiency of evidence in submissions of advanced therapies for marketing authorization and to benchmark them against more established biological products, we conducted a matched comparison of the regulatory submissions between ATMPs and other biologicals. We applied a quantitative assessment of the regulatory objections and divergence from the expected data requirements as indicators of sufficiency of evidence and regulatory flexibilty, respectively. Our results demonstrated that product manufacturing was challenging regardless of the product type. Advanced therapies displayed critical deficiencies in the submitted clinical data. The submitted non-clinical data packages benefited the most from regulatory flexibility. Additionally, ATMP developers need to comply with more commitments in the post-approval phase, which might add pressure on market performance. Mitigating such observed deficiencies in future product development, may leverage their potential for market success.

Report of the international conference on regulatory endeavors towards the sound development of human cell therapy products.

The regulation of human cell therapy products is a key factor in their development and use to treat human diseases. In that regard, there is a recognized need for a global effort to develop a set of common principles that may serve to facilitate a convergence of regulatory approaches to ensure the smooth and efficient evaluation of products. This conference, with experts from regulatory agencies, industry, and academia, contributed to the process of developing such a document. Elements that could form a minimum consensus package of requirements for evaluating human cell therapy products were the overall focus of the conference. The important regulatory considerations that are unique to human cell therapy products were highlighted. Sessions addressed specific points that are different from those of traditional biological/biotechnological protein products. Panel discussions complemented the presentations. The conference concluded that most of the current regulatory framework is appropriate for cell therapy, but there are some areas where the application of the requirements for traditional biologicals is inappropriate. In addition, it was agreed that there is a need for international consensus on core regulatory elements, and that one of the major international organizations should take the lead in formulating such a consensus document.

Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards.

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.

Do human leukocyte antigen-typed cellular therapeutics based on induced pluripotent stem cells make commercial sense?

The promise of off-the-shelf cellular therapeutics (CTPs) based on allogeneic induced pluripotent stem cells (iPSCs) may be hindered by alloimmunity, leading many to suggest that such products could be based on a series of human leukocyte antigen (HLA)-typed iPSC lines allowing at least some degree of tissue matching. While based on sound scientific principles, this suggestion presupposes that other immune responses will not be limiting. Technically this approach would present a number of major challenges, the first being the development of a suitably reliable reprogramming method amenable to validation that results in highly consistent iPSC lines. Further, the resulting array of HLA-typed iPSCs would need to be shown to be capable of being manufactured into the same CTP and exhibit comparable quality, safety, and efficacy. When the enormities of these challenges are laid out, it becomes apparent that the manufacturing and product development challenges would be unprecedented. Given the uncertainties and lack of clinical experience with iPSC-based CTPs at this time, the financial costs and commercial risks do not appear to be acceptable.

Reference materials for cellular therapeutics.

The development of cellular therapeutics (CTP) takes place over many years, and, where successful, the developer will anticipate the product to be in clinical use for decades. Successful demonstration of manufacturing and quality consistency is dependent on the use of complex analytical methods; thus, the risk of process and method drift over time is high. The use of reference materials (RM) is an established scientific principle and as such also a regulatory requirement. The various uses of RM in the context of CTP manufacturing and quality are discussed, along with why they are needed for living cell products and the analytical methods applied to them. Relatively few consensus RM exist that are suitable for even common methods used by CTP developers, such as flow cytometry. Others have also identified this need and made proposals; however, great care will be needed to ensure any consensus RM that result are fit for purpose. Such consensus RM probably will need to be applied to specific standardized methods, and the idea that a single RM can have wide applicability is challenged. Written standards, including standardized methods, together with appropriate measurement RM are probably the most appropriate way to define specific starting cell types. The characteristics of a specific CTP will to some degree deviate from those of the starting cells; consequently, a product RM remains the best solution where feasible. Each CTP developer must consider how and what types of RM should be used to ensure the reliability of their own analytical measurements.

The implementation of novel collaborative structures for the identification and resolution of barriers to pluripotent stem cell translation.

Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing-of expertise and/or funding-has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.

Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry.

The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.

Regulating interface science healthcare products: myths and uncertainties.

Whenever new technology emerges it brings with it concerns and uncertainties about whether or how it will need to be regulated, particularly when it is applied to human healthcare. Drawing on the recent history in the European Union (EU) of the regulation of cell-based medicinal products, and in particular tissue-engineered products, this paper explores the myths that persist around their regulation and speculates on whether the existing regulatory landscape in the EU is flexible enough to incorporate nanotechnology and other new technologies into healthcare products. By untangling these myths a number of clear conclusions are revealed that, when considered in the context of risk-benefit, make it clear that what hinders the uptake of new technology is not regulatory process but basic science.

Development and functional capacity of transplanted rat metanephroi.

BACKGROUND: Transplantation of embryonic kidneys (metanephroi) offers a potential solution to the problem of kidney donor shortage. The aim of this study was to characterise the haemodynamic capacity of transplanted rat metanephroi and to determine the number and maturity of the tubules. METHODS: Metanephroi from E15 Lewis rat embryos were transplanted adjacent to the abdominal aorta of uninephrectomised adult female syngeneic Lewis rats. Twenty-one days later, a single metanephros ureter was anastomosed to the host's urinary system. Three months later animals were prepared for standard clearance measurements. RESULTS: Effective renal blood flow (149 +/- 33 microl min(-1) per g kidney weight) and glomerular filtration rate (17 +/- 9 microl min(-1) per g kidney weight), standardised to kidney weight, were significantly lower in transplanted metanephroi compared with control adult kidneys (P < 0.001); renal vascular resistance (934 +/- 209 mmHg ml min(-1) per g kidney weight) was significantly higher (P < 0.001). Nephron number in transplanted metanephroi was significantly greater than that of E21 kidneys (P < 0.01) but lower than that of postnatal day (PND) 1 kidneys (P < 0.001). Angiotensin II type 2 receptor mRNA expression, a marker of nephrogenesis, was markedly reduced in metanephroi. Aquaporins 1 and 2, epithelial Na channel and Na-K-2Cl cotransporter type 2 mRNA and protein were expressed in transplanted metanephroi; the urea transporters-A1, 2 and 3 were absent. Vascular markers (alpha-smooth muscle actin and CD31) were identified in metanephroi but their expression did not differ from that of E21 and PND 1 kidneys. CONCLUSIONS: This study shows that metanephroi continue to develop post-transplantation but only reach a stage of development equivalent to that of a normal rat kidney at birth.

Increasing renal mass improves survival in anephric rats following metanephros transplantation.

Renal failure and end-stage renal disease are prevalent diseases associated with high levels of morbidity and mortality, the preferred treatment for which is kidney transplantation. However, the gulf between supply and demand for kidneys remains high and is growing every year. A potential alternative to the transplantation of mature adult kidneys is the transplantation of the developing renal primordium, the metanephros. It has been shown previously, in rodent models, that transplantation of a metanephros can provide renal function capable of prolonging survival in anephric animals. The aim of the present study was to determine whether increasing the mass of transplanted tissue can prolong survival further. Embryonic day 15 rat metanephroi were transplanted into the peritoneum of anaesthetized adult rat recipients. Twenty-one days later, the transplanted metanephroi were anastomosed to the recipient's urinary system, and 35 days following anastomosis the animal's native renal mass was removed. Survival times and composition of the excreted fluid were determined. Rats with single metanephros transplants survived 29 h longer than anephric controls (P < 0.001); animals with two metanephroi survived 44 h longer (P < 0.001). A dilute urine was formed, with low concentrations of sodium, potassium and urea; potassium and urea concentrations were elevated in terminal serum samples, but sodium concentration and osmolality were comparable to control values. These data show that survival time is proportional to the mass of functional renal tissue. While transplanted metanephroi cannot currently provide life-sustaining renal function, this approach may have therapeutic benefit in the future.