Introduction
The use of cells instead of molecules as medicines has opened new opportunities to treat diseases that arise from aberrant cell functionality. For example, cell therapy drug products can treat patients who have lost a cell type or who have gained an undesired cell type, such as a tumour cell. Successful treatment is dependent on the medicine containing living, functional cells of the desired type, at the correct dose, and free of contamination. While the complex nature of cellular medicines cells enables novel therapeutic modes of action to be exploited, this complexity conspires to make characterisation of the drug product and associated manufacturing processes an imprecise activity. This contrasts with the situation faced by molecular medicines, where essentially complete knowledge of the drug product structure and composition is attainable. Two consequences follow from this. First, it is difficult to assess the batch to-batch consistency of cell therapy manufacturing processes because only a limited set of the many potential product attributes are measured during release testing. Second, it is extremely challenging to identify structural and functional properties of cellular medicines that correlate with clinical safety and clinical outcomes (referred to as critical quality attributes or CQAs). Both of these challenges are addressed by performing extensive analytical characterisation during product development.
In this article, I discuss the analytical approaches we are taking at Cellistic to develop and characterise allogeneic induced pluripotency stem cell (iPS cell)-based medicines during their manufacture. Much of the characterisation performed on iPS-cell-derived cell therapy products is similar to other types of cell therapy product, such as the measurement of cell viability, testing for microbial contamination, and development of potency assays. I will focus here on analytical characterisation that is particularly relevant for iPS cell-derived products.
The link between product CQAs and manufacturing processes
The type of characterisation required for an iPS cell-derived drug product is ultimately derived from a list of the known, and suspected, CQAs. This tentative list is based on the target product profile as well as the current understanding of relevant pre-clinical, CMC, and clinical data. Importantly, CQAs refer not only to desired structural and functional features of the active ingredient itself but also to properties that arise from the manufacturing process, including, for example, the presence of contaminants and impurities. The novel processes that are used to generate iPS cell-derived drug products are therefore a source of new types of potential CQAs that require investigation. Several examples of iPS cell process-linked CQAs are discussed next.
Characterisation of iPS cell differentiation by flow cytometry
In vitro expansion and differentiation of iPS cells into a desired cell type is achieved by the controlled application of cytokines and other media components at defined time points during cell culture. While cell expansion is relatively straightforward to monitor (for example by automated cell counting), the characterisation of cell differentiation is more elaborate. Multicolor flow cytometry is the method of choice to measure the progress of iPS cell differentiation, revealed through the ordered appearance of different surface and intracellular markers. These markers are used to identify, characterize, and quantify on-path process intermediates as well as off-path cellular impurities. The initial choice of markers is guided both by in vivo expression data from primary cells, as well as anticipated in vitro expression patterns based on the process design. Typically, several panels containing 5-10 markers are used to monitor iPS cell processes during process development, where assay robustness and time to develop flow panels are critical factors. Data generated during process development is invaluable for identifying correlations between marker expression patterns and successful process outcomes. Once a correlation is established, reduced panels are implemented as in-process controls during manufacturing or as release tests in quality control.
Monitoring of genetic aberrations
The ability of iPS cells to proliferate indefinitely offers an attractive route to large-scale manufacturing of cell therapies. However, reprogramming, gene editing, and prolonged passaging of iPS cells comes with a risk of introducing genetic aberrations. The study of genetic changes promoted by these processes is an active area of research and have revealed that structural changes occur from the level of point mutations and indels to larger structural variants, such as changes in chromosome number. Since genetic changes have the potential to alter the efficacy or safety profile of a cell therapy, notably by increasing the risk of tumorigenicity, screening of iPS cells during clone selection and of the drug product at the release stage is required.
Fortunately, a number of analytical methods have been developed that enable screening of genetic aberrations. These include next-generation sequencing and PCR approaches that can detect point mutations and changes in copy number. Larger structural variants can be detected using image-based techniques such as karyology and optical genome mapping. Importantly, a suite of different methods is required to fully evaluate the presence of genetic aberrations in an iPC cell-derived product because each technique is only able to detect a subset of genetic aberration and with a defined sensitivity. For example, karyology is able to detect gross changes in condensed metaphase chromosomes that involve regions of DNA longer than 5 Mb and requires an analysis of 30 cells at metaphase to exclude 10% mosaicism (that is, to exclude an impurity level of 10-100% aberrant cells). Indeed, development of more sensitive assays to detect genetic aberrations is an active area of research in this field.
While detection of genetic aberration is relatively straightforward, a bigger challenge is to determine whether a genetic aberration, once detected, actually represents a risk for patient safety or drug efficacy. Some changes are known to represent a tumorigenicity risk (e.g., point mutation in the TP53 tumour suppressor gene) and are included in standard screening panels. However, most genetic aberrations reported in the literature have no known impact on cell function or patient safety. This risk can be mitigated by performing in vitro or in vivo tumorigenicity testing of the drug product (for example, cell proliferation assays), by including safety-switches in the drug product, and by long-term monitoring for clonal expansion in patients post-treatment.
Detection of residual iPS cells
Undifferentiated iPS cells have the ability form rare tumours called teratomas if injected in immunocompromised animals. The presence of residual iPS cells in the drug product represents an additional risk that is mitigated by analytical characterisation of the drug product. The minimum number of iPS cells required to form teratomas in patients is unknown, and so effort has again been expended on the development of sensitive assays that will allow residual iPS cell detection at different dose levels. Several RT PCR-based methods have bene reported in the literature with sensitivities of 0.001- 0.0001 % (i.e., able to detect a single iPS cell in a sample containing between 0.1 - 1 million cells of drug product). A critical step in the development of these RT-PCR approaches is to identify a target RNA sequence (either coding or non-coding) that is only present in the iPS cell line but absent from the drug product. RNA expression databases are used to guide the selection of specific and highly expressed sequences. A complementary approach to identify function residual iPS cells is to culture the drug product under conditions that favour the expansion of residual iPS cells and such assays show comparable or even better sensitivity.
Concluding remarks
Like other cell therapies, drug product characterisation forms an important part in the control strategy of iPS cell-derived cell therapy products. The unique reprogramming and differentiation processes of iPS cell medicines that enable reduced costs and wider access to cell therapies also introduce novel potential CQAs. Characterization of these CQAs during process development using the analytical approaches described here affords a route to ensuring patients receive safe and efficacious treatments with these pioneering medicines.