Q&A highlights: Virtual Symposium: New GMP iPSC lines and improved differentiation workflows for cell therapy

With regard to iPSCs – given their high proliferation rates and self-renewal capability, we think that banking multiple equivalent stocks with relatively few cells (in the range of millions) may be sufficient for many applications. This will require a short expansion phase after recovery and before initiating differentiation, but this may be acceptable and strategic. Conversion into some cell types like cardiomyocytes will, however, require large numbers of input cells, so yes, these approaches may benefit from iPSC upscaling and banking of larger quantities of cells. However, the full compatibility of different iPSC upscaling methodologies with specific differentiation protocols is yet to be shown in many of the relevant cases.

Regarding differentiated cells, the degree and demand for upscaling will critically depend on the respective patient doses. For example, the number of retinal pigment epithelial cells per patient for treating advanced macular degeneration will be significantly lower than the number of iPSC-derived CAR-NK cells in cancer immunotherapy patients. In this regard, we think our novel approach to accomplishing substantial cell expansion upon NK cell differentiation in suspension culture forms a viable basis for upscaling this important lineage.

While there is no single standard protocol for improving efficiency, in our experience, the initial steps of differentiation are critical. This is where the cells are most prone to get on the wrong track. This also implies that the starting state of the iPSCs must be tightly controlled. For instance, if you aim for >95% differentiation efficiency into cardiomyocytes, >95% of your iPSCs at the beginning of differentiation must be in a fully undifferentiated and responsive state. Therefore, as a first step, we attempt to tightly control and standardize our iPSC cultures, including the starting day and time, feeding schedule, seeding densities, confluency at harvest, etc. It is equally crucial to closely monitor the specific activities and integrity of all components of the differentiation media, as factor concentrations may have – in many cases – a profound impact on differentiation outcomes. Hence, when optimizing differentiation protocols, we tend to titrate all active components, optimize media change intervals, cell densities, etc. The devil is in the details. We see, though, that being passionate about details and aiming to achieve full control over the system pays off in the long term. Many of these aspects are actually implied in good manufacturing practice (GMP) procedures.

Regarding our differentiation workflows, as well as genetic manipulation of human iPSCs, our approaches were designed to avoid the need for purification steps altogether. However, some things can be done to help the process. In gene editing procedures, for instance, we always have a single cell seeding step, followed by clone analysis. As we only edit iPSCs followed by single-cell cloning, we currently do not use any purification steps. Differentiated cell types will always be derived from iPSCs, where the editing takes place.

Actions to consider during purification include:

1. You could create replica plates (when working with clones in 96-well plates) for in-process controls for screenings using flow cytometry (easiest for surface gene modifications).
2. Others knock in genes by tagging essential genes, such as the SLEEK approach by Editas. Here, indel creation disrupts an essential gene, leading to cell death, unless the HDR template is incorporated correctly. This enables selection without the use of an additional marker but may potentially affect endogenous gene function. Also, the gene must be expressed at the iPCs stage, as well as in the cell type used in downstream applications.
3. Some approaches use a piggyBac transposon technology, including one round of positive selection, marker excision and one round of negative selection, resulting in seamless editing if designed correctly. However, this approach may not be the best option in a GMP environment.

Different systems have been shown to be compatible with iPSC expansion, like the stirred tanks and the more recent Vertical Wheel Bioreactors. Stirred tanks can be coupled with perfusion systems, allowing controlled gas and nutrients throughout the culture. Using these systems iPSCs can expand and reach a density of 10⁷ cells/ml. On the other hand, vertical wheel bioreactors are more simple and economical systems that show a reduced shear stress, but due to the lack of perfusion, the cell expansion can be limited to 10⁶ cells/ml. However, this system is compatible with microcarriers, allowing those limitations to be overcome.

Not to a significant degree. A given iPSC clone tends to be more stable and consistent from batch to batch than, for instance, different iPSC lines to one another. Overall, we consider the pluripotent stem cell state a natural and stable one that essentially all integration-free lines may acquire within several passages, given appropriate culture conditions.

The administration of immune cells will most likely depend on the cancer type. While hematologic malignancies will likely be treated by a systemic or tumor-targeted intravenous injection of immune cells, solid tumors could additionally be treated with a local, intratumoral injection of immune cells. However, these decisions are governed by the corresponding innovators and clinicians and their therapeutic strategies.

Yes, Catalent has a validated GMP process reprogramming CD34+ cells to iPSCs by transfection of episomal vectors. The transfection of episomal vectors (plasmids) triggers an epigenetic conversion from CD34+ cells to iPSCs without genetic integration. Adherent colonies with pluripotent stem cell-like morphology are then isolated and expanded as distinct candidate clones cultured in a GMP-grade iPSC medium. Passaging is continued with a selected clone/line, and once vector clearance is confirmed, the clone is expanded and cryopreserved as a GMP master cell bank.

The protocol for gene editing is currently optimized in our research and development (R&D) department. However, it is designed to be compatible with GMP requirements from a manufacturing perspective and only utilizes equipment that can readily be used in a clean room setting. While certain quality control assays, especially with regard to off-targets, are not technically GMP certified, we tend to employ assays commonly used and accepted in the cell therapy field.

In order to achieve a density of 10⁷ cells/ml, the cell culture conditions have to be closely monitored and controlled, like nutrients, pH, temperature and dissolved oxygen. Currently, only stirred tank systems have been shown to provide optimal cell culture conditions, achieving a density of 10⁷ cells/ml of iPSC in suspension.

In terms of adherent culture, the Quantum hollow fiber bioreactor has been shown to almost reach 10⁷ iPSCs/ml densities (690 x10⁶ in 100ml) in a closed, perfusion-based system, equating to 14-fold expansion in under 7 days [1]. This bioreactor provides 21,000 square centimeters of surface area, equivalent to almost 100 T225 flasks.

This is certainly dictated by the respective process and cell lineage. Non-invasive checks are currently confined to assessing the morphology of cells grown or differentiated in 2D. Most other assays rely on taking representative samples to be subjected to flow cytometry analysis and similar readouts.

Yes, Catalent offers both R&D grade and GMP grade iPSCs. The R&D stocks are directly derived from the GMP ones using technically equivalent culture conditions, albeit under R&D conditions, and are genetically identical to the GMP cells apart from having a few additional passages. We offer readily available GMP iPSC cell lines from EU and US donors with a commercial license. Two HLA-homozygous EU lines are currently available as fully released GMP cells. One more line of EU origin is being quality-controlled for GMP release and features compliance with FDA and EMA (and PDMA) donor eligibility requirements. Two additional lines derived from US donors are available for testing under R&D conditions, and we expect QC release of the underlying GMP banks by Q1 of 2024.