Manufacturing with pluripotent stem cells (PSConf 2021): Key issues for future research and development

Abstract Human pluripotent stem cells (hPSC) have the capability to deliver novel cell‐based medicines that could transform medical treatments for a wide range of diseases including age‐related degenerative disorders and traumatic injury. In spite of significant investment in this area, due to the novel nature of these hPSC‐based medicines, there are challenges in almost all aspects of their manufacturing including bioprocessing, characterization and delivery. The Chinese Academy of Sciences and the Chinese Society for Stem Cell Research have collaborated to create a new discussion forum called PSConf 2021 (Pluripotent Stem Cell Conference 2021), intended to promote exchanges in communication on cutting‐edge developments and international coordination in hPSC manufacturing. The PSConf 2021 addressed crucial topics in stem cell‐based manufacturing, including stem cell differentiation, culture scale‐up, product formulation and release. This report summarizes the proceedings and conclusions from the discussion sessions, and it is accompanied by publication of individual papers from the speakers at the PSConf 2021. Significance Statement The PSConf 2021 meeting has brought together speakers and delegates from more than 20 countries in an informal discussion forum focusing on the manufacture of cell‐based medicines using hPSCs. The conference discussion sessions enabled an open exchange of information on the latest developments, ideas on key challenges and their potential solutions. It also captured the experiences and lessons learnt by professionals who had been in the field from the earliest applications of human embryonic stem cells, and presented a diverse range of new potential pluripotent stem cell‐based medicines that are now under development, with some already in clinical trials.

been huge international investment in basic research to explore their potential in improving our understanding of cell biology and early development, in vitro diagnostic systems for drug discovery and safety testing, and regenerative cell-based medicines. In more recent years, the potential for real applications of human pluripotent stem cells (hPSCs) in some of these fields has begun to be realised 3,4 and, in particular, independent monitoring of the development of hPSC clinical applications indicates that there are now more than 100 clinical trials for products manufactured using hPSCs. 5 The Institute of Zoology (IOZ), Chinese Academy of Sciences  Dr Alex Zhang (Zephyrm Biotechnologies, China) presented an overview of the key issues from the perspective of an innovative pharmaceutical company and now an early independent developer of cell-based medicines. He began by identifying top-priority challenges that were particular to a cell-based medicine: • Safety in terms of in vivo distribution, tumorigenicity and immunogenicity/immunotoxicology, • Efficacy in terms of generating the appropriate cell type for the intended indication, mechanism of action and obtaining the appropriate dose for patients, • Establishment of a robust Quality Management System to assure reproducible products, with special attention to cell heterogeneity and sustained characteristics after industrial scale production.
Dr Zhang compared the manufacturing process of monoclonal antibodies with hPSC-based manufacturing and indicated the common requirements of establishing a stable manufacturing cell line, cell banking, culture expansion, purification of hPSC-derivative progenies and formulation of the final product. However, he noted the significantly greater complexity of bioprocessing hPSCs. Whilst he acknowledged that many labs could learn the hPSC culture technology, it was nevertheless challenging to translate and scale-up the technology in a manufacturing environment. Furthermore, hPSCs were prone to appearance of genetic variants which may have significant yet hith- In order to generate robust and reproducible bioprocessing, Dr Zhang explained how he had used process analytical tools to identify those Critical Manufacturing Attributes (CMA), including requirements for critical reagents (e.g., nutrients/media, growth factors, microcarriers, cell seeding density) and process CMAs (e.g., cytokine profiles, glucose levels). This enables system modelling based on such process CMAs and Critical Process Parameters (CPP), such as the metabolic profiles and rate of changes in pH. In conclusion, Dr Zhang emphasized the crucial importance of high quality and informative Critical Quality Attributes (CQA) for the final product, including parameters for phenotypic identity, purity and potency. He saw the development of large-scale bioreactor systems and the maintenance of genetic and phenotypic stability throughout the manufacturing process, as the most significant challenges remaining for the clinical utility of M-cell products. This topic has also been explored in greater detail by Zhang and Stacey (2022). 6 Dr Cedric Ghevaert (Wellcome Trust-MRC Cambridge Stem Cell Institute and NHS Blood and Transplant, UK) presented on the development of a manufacturing process for a clinical trial based on hESCderived megakaryocytes being developed as part of the UK Regenerative Medicine Programme (https://www.ukrmp.org.uk/home-extended/ mrc/). He explained that whilst this work involved the development of a cell type that had been used for many years, the use of a new and substantially different bioprocessing technology would require an almost complete rethinking for the whole process of product delivery and use.
Dr Ghevaert outlined the complex multistage manufacturing process, with key intermediate active ingredient stages of megakaryocyteerythroid progenitors, megakaryocytes and platelets requiring a 100-day process of directed differentiation based on a predefined sequence of environmental cues, including cytokines and feeder cells. He also described the additional work required to source raw materials of suitable quality for manufacturing and the subsequent optimisation of culture conditions. He explained the significant benefits in the optimisation of platelet yield that this process had delivered. 7 He also emphasized that understanding the culture and differentiation process through process analytical tools, with a focus on those stages that were most challenging to develop into a GMP environment, had been key to progress towards a final manufacturing process. 8 Dr Ghevaert outlined the significant work involved in the development of new potency assays including an in vitro thrombus formation and a laser-induced injury model in NSG mice. 6,8 A major issue that the whole hPSC manufacturing field is facing today, is the selection of appropriate manufacturing cell lines. Dr Ghevaert explained the significant diversity that the UKRMP programme had discovered in the differentiation characteristics of different hESC lines. He concluded by stating that significant challenges remained in scale-up, cost-science-based safety and quality standards, and achieving acceptability and adoption by healthcare providers. One advantage of hPSC-derived platelet products was that they were generally considered to be more like traditional protein biologics, and not a cell therapy, as they were small cell fragments that were not nucleated.
However, this regulatory perspective had also brought a focus on assuring a very high degree of product purity. To conclude, Dr Ghevaert reported early work for future developments to transition from 2D to 3D manufacturing processes using 'sponges' containing perfusion bioreactors and he also saw great possibilities for 'supercharged platelets' modified by TALENS gene editing to increase the levels of von Willibrand factor expression and product clotting performance. 6,8 It was clear from Dr Ghevaert's presentation that even hPSC-derived products that appear quite simple, such as platelets, still require the resolution of complex biological challenges to deliver a reliable manufacturing process. She also urged product developers to plan for suitably scaled production that would meet the patient population needs at a cost that would enable commercial development.
Dr Kapil Bharti (National Eye Institute, National Institutes for Health, USA) reflected on a key challenge in his programme to manufacture a retinal pigmented epithelium (RPE) product using iPSC lines derived from the patients, which was that each product was derived using a different starting material. He reported that this issue had meant that IND-enabling studies for autologous products in the US require manufacturing process validation before each clinical product can be manufactured, whereas this was not the case for allogenic under xeno-free conditions, 9 and they had developed approaches for derivation, expansion and differentiation of hPSCs all in 3D suspension format, so that it would be more amenable to large scale operations. 10 These yielded reproducible batches of hESC clusters that could be converted to neural cell spheroids. They showed that 3D dynamic culture technology, such as the use of stirred tank bioreactors at various stages of manufacture, yielded high cell numbers that were sufficient for multiple clinical doses. In their ongoing research, Prof. Reubinoff and Ms. Tannenbaum presented various options for suspension bioreactors, and concluded that a vertical stirred tank system (PBS Biotech, Inc., Vertical-Wheel™ Bioreactor) provided uniform distribution of hydrodynamic forces, thus, reducing shear stress and yielded cell aggregates of uniform size, which sustained a diploid karyotype and pluripotent characteristics. Furthermore, this system gave significantly higher cell number yields compared to typical horizontalblade spinner-type stirred tanks systems. 11 Their work had also revealed the key factors in successful high yield suspension culture, that include selected seeding density, careful timing of media changes to control nutrient and metabolite levels, and selection of suitable tools to increase culture attachment surface area (e.g., microbeads, aggregates, hydrogels). Prof. Reubinoff and Ms. Tannenbaum detail, which is published in tandem with this meeting report. 12

| ANALYTICAL METHODS AND RELEASE
Prof Roger Barker (University of Cambridge and Addenbrookes hospital Brain Repair Centre, UK) led the discussion by explaining the importance of selecting a disease and a relevant patient group within that disease entity, that would be likely to benefit most from a cellbased therapeutic approach. In the case of Parkinson's Disease, he described the two approaches; (i) to actually repair the dopaminergic cellular network (using cell or gene therapy) or (ii) arrest the disease process or slow it down (e.g., anti-inflammatory treatments, alphasynuclein based treatments). He described his work, supported by an EU consortium, focusing on allogenic cell replacement with hPSC ventral mid-brain progenitor cells as the STEM-PD product to replace midbrain A9 dopaminergic neurones. He emphasized the importance of selecting a suitable production cell line, by assessing its traceability and the whole process of its development. Dr Barker also reviewed the pros and cons of hESC-versus iPSC-lines and concluded that whilst iPSCs had a less controversial ethical status and held the promise of HLA-matched therapy, they also carried more concerns regarding tumorigenicity and long-term patient safety given how they are generated. He outlined the importance of reagents and protocols that met the requirements of GMP manufacturing and the extensive GLPaccredited testing at production cell bank and final product levels that were essential to meet regulatory acceptability. Prof. Barker also reviewed the extensive work that had been needed for bioprocessing protocol development and the in vivo and in vitro preclinical studies which had taken 6 years to complete. He looked forward to the conclusion of the STEM-PD clinical trial programme that was currently moving to deliver a clinical trial in 2022.
Following Prof Barker's talk, discussion with delegates included consideration of the need for CE marking for devices in the EU, which may include certain equipment, reagents and testing systems used for hPSC manufacturing. It was clear that CE mark approval status would be required in the EU for such products used after the clinical trial stage and may need considerable effort and cost to achieve approval. The variation in product purity was also discussed and may require careful purification of therapeutic cells. Prof. Barker said that the culture system his group was using selected against residual stem cell populations. He also explained that in his collaboration with Prof. Malin Parmer (Lund University, Sweden), the Lund group had qualified the purity of the final product using antibody and molecular analysis of cell populations.
The issue of preclinical assessment of implanted cell biodistribution was addressed in detail by Prof. Patricia Murray (University of Liverpool, UK) whose centre had utilized a portfolio of novel imaging approaches. 13 In this presentation, Prof. Murray focused on noninvasive imaging techniques for studies of MSCs and hPSC-based products. In order for biodistribution studies to yield useful data, Prof.
Murray considered that it was important to develop techniques that would enable better understanding of cell viability, but also the proliferative state and differentiation status. This would hopefully enable verification that therapeutic cells can access and populate the intended target site, and exclude potentially hazardous distribution and accumulation of cells at unintended sites. Tracking cells in this way would hopefully enable an important understanding of safety, efficacy and mode of action of the cell-based medicine. Prof. Murray emphasized that this knowledge is key to determination of the risk-benefit ratio of the therapeutic, and facilitate optimisation of product safety and efficacy. She also described a model for whole body biodistribution studies in mice using intra-cardiac inoculation of mesenchymal stromal cells that were genetically modified to express firefly luciferase and ZsGreen reporters. These studies had shown that bioluminescence was highly sensitive and gave data on cell viability and proliferation, but suffered from poor spatial resolution and yielded only planar images. Prof. Murray also overviewed other work using magnetic resonance imaging to visualize mesenchymal stromal cells labelled with ironoxide particles, which gave high spatial resolution that allowed analysis of intra-organ biodistribution, but did not indicate cell viability. She reported a recent collaboration with Prof. Roger Barker at the University of Cambridge, to use a combination of bioluminescence and iron-oxide labelling tracking methods followed by histological investigation to assess the fate of hESC-derived dopaminergic neuron progenitors in rats. 13 They are now assessing the use of materials that enable sustained release of specific growth factors such as GDNF and BDNF (PODS ® , developed by the UK company Cell Guidance Systems), to explore their ability to promote the viability and differentiation of dopaminergic progenitors following implantation.
In the discussion following Prof. Murray's presentation, she was asked about the availability of the PODS ® that enabled sustained release of specific growth factors. Prof. Murray said that these were commercially available from Cell Guidance Systems. Delegates also asked about the toxicity of the iron-oxide particles to dopaminergic neurones. Prof. Murray explained that iron-induced toxicity had not been observed at the levels used in mouse models. She added that the primary purpose of these experiments was to optimize and validate the safety of the dopaminergic neurone biodistribution for later human use without iron-oxide labels. Dr Kapil Bharti (National Eye Institute, NIH, USA) described the high degree of complexity in the manufacture of his autologous retinal pigmented epithelial product for treatment of age-AMD. It involved many reagents (including numerous growth factors), multiple production processes over a total production process time of 164 days. In the case of the NIH study, it had been necessary to acquire significant experience with more than 100 differentiation runs to achieve a reproducible manufacturing process where batches reliably met acceptance criteria. Crucial early in-process controls included selection of suitable iPSC clones and maintenance of sterility of cultures. It was also vital to have highly trained staff to avoid wasted time due to contaminations or selection of poor-quality clones. The lack of predictability for iPSC suitability using current in vitro pluripotency assays meant that for each donor 12 clones were established to provide adequate back up clones to avoid the need for repeated reprogramming. Other important inprocess quality control checks included application of HLA-typing at various stages to exclude any cross-contaminated cultures and therefore non-autologous product. Dr Bharti went on to summarize the key challenges at the reprogramming (assuring robust sample traceability, need for predictive assays for pluripotency, lack of consistency in colony selection and lack of closed processes to avoid contamination) and product release stages (need to release each batch of product, critical timing between product availability and patient treatment, sterility testing results). In the light of these numerous challenges, Dr Bharti stated that it was important to establish key go/no-go steps to avoid wasted time and resources on product that was not developing appropriately.
He went on to explain the complex process of validating his autologous iPSC cell therapy IND application to progress to Phase I/IIa trial which had required demonstration of consistent production with three clones from each of three iPSC donors. The release criteria established for clinical trial included flow cytometry for cell purity, quantified cell morphology assessment, RPE-specific gene expression, cytokine excretion (i.e., VEGF and PDGF) and phagocytosis activity. Dr Bharti also reported efforts of his group to increase the cost-effectiveness of autologous iPSC therapy manufacture. In particular he had been pilottesting automated reprogramming using the Artitell (Cellino) colony picking device, which used AI-based characterization and laser-based elimination of poor-quality iPSC colonies whilst holding individual batches of patient cells in a closed system until cell lines had been established. Final colony selection was also confirmed following Score-card™ analysis of pluripotent potential. Dr Bharti saw such automated AI-based approaches as crucial to enabling cost-effective autologous iPSC therapies.
In Q&A, delegates expressed great interest in accessing the artificial intelligence validation tools Dr Bharti had described. He reported that there were plans to make the software available via a Cloud-based system. report, a more detailed consideration of the issues. 12,29,30,33 In open discussion with Prof. Isasi and Dr. Peng, delegates asked about the degree to which governance for individual tissues from donors fell under local or regional/national authorization. Prof. Isasi responded that this depended on whether the tissue in question was somatic or embryonic/foetal, but concluded that national law would dominate. However, she also emphasized that it was valuable to consider international regulations that may impact on stem cell products in the future. Furthermore, Prof. Isasi considered that professional best practice was especially important in areas where existing regulation was lacking. Dr. Peng added that in China two documents regu- A further concern amongst the delegates was the major challenges in assessing variation in ethical requirements in different jurisdictions. Prof. Isasi said this is fertile ground for progress especially in relation to provenance of cell lines and approval processes, where there was need for more concentrated efforts. She went on to explain that the barriers were not too significant but would require crossborder understanding. Dr. Peng stated that adopting appropriate forms of communication is a key challenge in this area, including the need to share regulations and laws. Prof. Isasi also emphasized that good science should begin with good policy and ethics. It was generally concluded that focusing too much on interjurisdictional regulatory and ethical differences is unhelpful and it is crucial to consider levels of harmonization that already exist. Overall, the requirements for fundamental protection of donors and research integrity are similar in different regions (see Appendix 3 reference). 20

| Bioprocessing and differentiation in manufacturing
• Understanding of cell line genetic stability and ability to differentiate into fully effective mature cell types.
• More knowledge required on cell system interactions to support identification of relevant Critical Process Parameters.
• Huge technical challenges in more complex and lengthy differentiation pathways that need measures to assure purity and reliability of product batches.
• Automation for well-controlled reproducible manufacturing processes.
• Bioreactor scale-up systems and cost-effective manufacturing processes will be critical to success as many hPSC-based products require significant expansion and product batches would need to exceed 10 9 therapeutic cells. Effective tools and protocols for biopreservation and recovery • Understanding of cell population interactions and impact of culture systems on cell performance.

| Product formulation and testing
• Mode of action is often not well understood at least at the start and it will be important to have greater understanding of how cells work in the respective disease environment.
• Safety of engineered cells • Relevant assays of tumorigenicity that are informative for patient safety • Validation of animal models and new tools to investigate biodistribution, cell function and safety.
• New research and validated tools to enable identification of relevant and effective CQAs.
• Investigation and validation of new biomarkers and functional assays as predictive indicators of efficacy.
• Quantitative evaluation of biomarkers and functionality • Artificial Intelligence systems will be needed to process large and complex data sets required for hPSC-based manufacturing.
• Potency assays for different therapeutic cell types.

| The need for standardization
• International co-ordination is crucial and could be most effective if directed at precompetitive aspects where there could be the broadest benefit. A number of key scientific topics were identified for ongoing cooperation between the groups and these included: • Standards relating to the generation of high-quality stem cells.
• Genetic stability and its relationship to the safety of cell-based medicines.
• Safety and efficacy of cell-based medicines derived from stem cells • Utility and interoperability of stem cell database systems.
• International regulatory issues and best practice especially in research governance and regulatory science to advance the development of stem cell therapy.
• Training initiatives for stem cell banking, characterization and bioprocessing.
• Stem cell ethics and data sharing.
In conclusion, the Bridging meeting partners agreed to promote the international coordination and co-operation by exchanging further information on their respective activities in the aforementioned areas and plan for follow up interactions to consider possible programmes of collaborative work.
In a personal summary, Dr. Andreas Kurtz (Fraunhofer-IBMT, Germany and Coordinator of hPSCreg) covered a range of issues he considered to be important for future hPSC-based therapies. In particular, he endorsed the need for international co-ordination which had been a key element in the development of the hPSCreg project and had been mentioned by workshop panellists. In relation to research ethics, he pointed to key similarities but also differences such as the implementation of social morality in regulations of the EU and China. In terms of scientific issues for hPSC-based medicines, Dr. Kurtz emphasized the importance of understanding the genetics of therapeutic cells.
The characterization of genetic variants and target cell standards would be important, including quality control/safety testing, cell identity and genetic/epigenetic tolerance for genotypes acceptable for clinical use.
He went on to outline areas requiring active coordination and development of best practices in research ethics, including: • Early and careful consideration of ethics issues to avoid problems in the mid-long term Finally Prof. Qi Zhou (CAS, China), the initiator of the PSConf 2021 meeting, concluded that the levels of scientific achievement presented at the meeting were very exciting and the discussions were of a very high quality. He was glad to see the collaborative spirit that delegates and speakers had brought to the meeting. Prof. Zhou also noted that the PSConf meeting series aims to bring global scientists, manufacturers, product developers and regulators together to brave the new challenges of hPSC translation in unison, and envision the future of hPSC translation together. Therefore, he hoped the meeting series will help to forge broader collaborations to advance the frontiers in this exciting field of stem cell and regenerative medicine.
He encouraged all delegates and speakers to further build upon the interactions which the meeting had initiated, and he looked forward to welcoming delegates from all countries to future meetings.