QC for iPSC-derived therapy
Published on: July 13, 2024
Human induced pluripotent stem cells (iPSCs) can be expanded and differentiated into committed pluripotent stem cells with relevant therapeutic potential. As a new source of starting material for cell therapies, iPSCs have demonstrated significant promise in regenerative and personalized medicine. However, iPSC-derived therapies come with numerous challenges particularly in cell stage identification, monitoring method selections during cell expansion, cell stability, and the quantity of the cells with therapeutic potentials in the final product during manufacturing. Although certain testing methods are widely accepted in the industry for iPSC release (see Figure 1), several issues remain:
Figure 1: Common tests for release and characterization. A) General compendial tests, including visual inspection, pH and osmolality test, endotoxin level determination and sterility to exclude microbial contamination (bacteria, fungus, mycoplasma and pre-existed human pathogens). B) Cell stage Identity and % cells with the capability of therapeutic potential for designated diseases. C) Safety of the stem cell reagents. D) Drug stability under long-term storage and transportation. (Source doi.org/10.1016/j.btre.2023.e00784). The quality assessment strategies for different iPSC, including committed pluripotent stem cells manufacturing, must be individually designed, developed, and performed based on the “Quality Attributes” of the projects prior to GMP manufacturing.
The design of flow cytometry panels depends on:
Starting materials
Pluripotent stem cells generated through reprogramming or established during expansion
Final differentiated cell therapy products
Detection of starting materials: Various tissues might require detecting a combination of “stem” cell specific cell surface, intracellular, and intranuclear markers, such as human umbilical cord blood, that can be used to create iPSCs. Common surface markers to identify stem cell potential include CD34 (hematopoietic stem cell and progenitor cell marker), CD9 (leukocyte antigen), CD133 (Prominin 1), CD30 (tumor necrosis factor receptor), CD200 (MRC OX-2 antigen), and CD38 (ADP-ribosyl cyclase).
Figure 2: Schematic diagram of the differentiation of iPSC cell pools generated from umbilical cord blood CD34+ cells into cardiomyocytes, T cells, and NK cells.
Detection of Embryonic Pluripotent Stem Cells: Some undifferentiated embryonic pluripotency markers such as intracellular transcription activation factors, OCT4, NANOG, and SOX2 and their relative expression ratio with other transcription factors are evaluated for cell stemness.
By spontaneous or induced differentiation, new committed stem cell specific transcription factors or cell markers might appear with cell proliferation. These new cell surface or intracellular markers might indicate the cell differentiation stage and their therapeutic potential for specific diseases. The ratio of undifferentiated residual iPSCs and committed differentiated cells in fixed stages must be defined.
The residual aberrant cells with rapid proliferation and cell drifting must be eliminated to an acceptable level prior to clinical application. Commonly used intracellular and cell surface markers for stage identification include TRA-1-60, Stage Specific Embryonic Antigen 4 (SSEA-4), TRA-1-81, and more than several dozens of stem cell related markers for committed differentiation identification. For example, SSEA-4 TRA-1-60 and TRA-1-81, which are detected later in the differentiation process without a differentiation commitment.
Similar stemness identification strategies can be carried out through molecular biological methods, such as RT-qPCR for specific stem cell factor expression and quantified by comparing the expression level of each target molecule with selected house-keeping genes. Both approaches are rapid detection assays and generally comparable
Detection of final cell therapy products: iPSCs can be differentiated into various therapeutic cells, such as therapeutic cardiomyocytes, T cells, NK cells, and B cells (see Figure 2). Testing for characterization and purity of these final products is essential in the release panel. For example, a flow cytometry panel for cardiomyocytes includes markers like cTnT, calcium channels, myosin chains, and GATA-4. Panels for T cells include lymphocyte markers like CD3, CD4, CD8, and CD56 (negative market), while NK cell panels include CD16 (FcγRIII), CD57, CD56+/CD3-, and NKG2C.
Detection for Stem cells (ESC or iPSC): Specific staining panels have been developed to assess the quality of starting materials. For example, monitor CD34+ cells from umbilical cord blood on day 4 of expansion, and evaluate the phenotypic characteristics of iPSC after reprogramming and expansion.
Furthermore, flow cytometry panels have been developed for various cell therapy products. For instance, insulin-secreting β cells are tested using panels that include markers like C-peptide, NKX6.1, PDX1, and MNX1.
Cell counting and viability assessment are critical for determining the number of viable cells especially in the context of final differentiated cell products. Viable cell density is a key parameter in CCV tests for the final differentiated cell product. For iPSC, the count of differentiated cells is also important for consistency in the initial feed and differentiation processes. Accurate measurement of viable cells during differentiation initiation, passage, expansion, harvest, and recovery from cryopreservation helps monitor and access the dosage of final differentiated cells.
However, iPSCs often form large colonies, which can make manual counting challenging due to overlapping cells. To address this issue, enzymatic, chemical, or mechanical separation methods are employed for continuous passaging. Automated cell counting technologies, such as NucleoCounter® NC-200, Vi-CELL Cell Viability Analyzer, and Cellometer Auto T4, have been developed to improve accuracy, homogeneity, and efficiency. Devices such as the NC-100 and NC-200 are effective for accessing viable cell numbers and viability in iPSC platforms.
Automated NucleoCounter NC-200 (Chemometec) uses acridine orange and propidium iodide, while Cellometer (Nexcelom) and Vi-CELL (Beckman Coulter) use Trypan Blue as a viability dye. These systems are suitable for counting specific cell types, with clustered cell counting protocols applied to iPSC.
Reprogramming clearance involves detecting residual plasmids, viral vectors, and transposons used during reprogramming, as well as residual “Yamanaka factors” (Oct4, Sox2, Klf4, c-Myc, and Lin28) after reprogramming.
EBNA/OriP plasmids are commonly used because they are non-integrating and can be removed from cells after passing. For safety and regulatory compliance, reliable methods are needed to quantify or confirm the complete removal of residual plasmids. TaqMan-based qPCR is a reliable method for detecting residual plasmids, offering high specificity and sensitivity for EBNA and OriP plasmids. However, fragments outside the EBNA/OriP region may not be detected.
As technology progresses, more sensitive methods such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS) are becoming available. ddPCR is more sensitive than qPCR, providing absolute quantification of nucleic acids by dividing samples into droplets, each undergoing PCR amplification. Analysis of the proportion of positive droplets allows for determination of concentration of target DNA templates. Although ddPCR can still miss residual plasmid fragments, using multiple primers can help detect different regions of the plasmid.
NGS can sequence all DNA or RNA in a sample, not just the EBNA/OriP region, making it a valuable tool for detecting residual reprogramming plasmids. However, whole-genome sequencing remains costly, with platforms like Illumina MiSEQ and ABI SOLiD available for commercial use.
Other impurity issues include biological additives during the cell expansion, such as protein -based colloid molecules, growth factors, cell debris, and oncogene activation, etc. These issues can be resolved individually by specific commercial assays. In addition, impurity from drug-self, such as unequal mitosis resulting in karyotype abnormalities can be resolved by karyotype analysis.
Master cell banks (MCBs) and allogeneic cell products for clinical use must be tested for viral contaminants, including retroviruses, adeno-associated viruses, and various animal and human viruses.
In retrovirus detection, the Product Enhanced Retroviral Test (PERT) is commonly used. PERT involves introducing samples to an RNA-based PCR system. If the sample contains retroviral reverse transcriptase, it will be detected through PCR amplification, indicating the presence of retroviral contamination. However, excessive DNA polymerase activity can interfere with detection, leading to potential false results. Two common PERT-based methods are QPERT and FPERT.
QPERT (Quantitative PCR Enhanced Reverse Transcriptase): It provides a means to quantify the level of retroviral reverse transcriptase present in a sample, offering insights into the extent of retroviral contamination. Since it monitors the entire process, initial false positives can be addressed appropriately.
FPERT (Fluorescent PCR Enhanced Reverse Transcriptase) is a qualitative method used to access RT activity during large-scale harvesting and in the final product to detect retroviral contamination. This method incorporates a fluorometric approach to enhance the sensitivity and accuracy of retroviral reverse transcriptase detection, aiding in the identification of retroviral contamination. Industry standards typically use FPERT results for product release and QPERT for process monitoring.