Clinical manufacturing of CAR-T cells for adoptive immunotherapy
– using the NucleoCounter® instruments
The NucleoCounter® NC-202™ and NucleoCounter® NC-200™ provide robust and accurate determination of cell count and viability of T cells during adoptive CAR-T cell manufacturing. The unique Via2-Cassette™ combines cell sampling, staining and counting chamber-loading into a single step, followed by automatic image acquisition and analysis by the NucleoCounter® for increased robustness.
Accurate detection of T cells, even in the presence of erythrocytes, beads or formulation reagents are facilitated by acridine orange (AO) and DAPI, which are already pre-loaded into the Via2-Cassette™. As a result, the NucleoCounter® system is ideal for adoptive CAR-T cell manufacturing.
Adoptive immunotherapies using engineered CAR-T cells
Monitoring the whole process from leukapheresis to the formulated product using the NucleoCounter® NC-202™
Figure 1: Monitoring the whole process from leukapheresis to the formulated product. With the NucleoCounter® NC-202™ it is easy to monitor all different steps of the purification, expansion and formulation of CAR-T cells using the same instrument to ensure precise and reliable results.
Easy and user-adaptable protocols allow performing cell counting directly from leukapheresis as well as from purified and expanded CAR-T cells using the same instrument. The Via2-Cassette™ is pre-loaded with the two dyes acridine orange (AO) and DAPI, avoiding some of the problems that lead to significant inaccuracies when doing bright field-based cell counting. For samples containing erythrocytes, pre-treatment with an erythrocyte lysis buffer eliminates the quenching effect of hemoglobin. The total cell count and the dead cell count are determined by staining with AO and DAPI, respectively, using the Via2-Cassette™. The analysis excludes cell fragments and artifacts like micelles as well as undersized events such as platelets, giving a highly accurate result. During isolation of T cells, magnetic beads may be used. These beads will have no influence at all on the cell counting with the NucleoCounter® NC-202™. In conclusion, the NucleoCounter® NC-202™ can be used for every step of the cell manufacturing process.
Application note (.PDF):
Superior data management and presentation
Figure 2: Superior data management and presentation using the accompanying NC-View™ (for NucleoCounter® NC-202™) or NucleoView™ (for NucleoCounter® NC-200™) software. The software allows for easy coupling between the obtained image and plots showing quantitative fluorescence for precise control of the analysis.
The NucleoView™ software allows visual inspection of the fluorescence image and the opportunity to verify the counting (Figure 2). Specific event populations can be selected in the scatter plots and examined visually to determine the validity of their inclusion or exclusion from the final counting results. The adapted protocol can be then saved for future measurements, increasing reproducibility.
The Nucleocounter® NC-202™ is calibrated and GMP-ready for 21 CFR Part 11
Figure 3: Automate and standardize PDF reports. The PDF reports ensure accurate documentation and verification of the obtained results by using electronic approvals and signatures.
The mounting success of CAR-T clinical trials now requires the development of GMP manufacturing of clinical-grade CAR-T cells in order to safely commercialize this promising personalized therapy. The NucleoCounter® NC-202™ can be easily implemented in GMP and 21 CFR Part 11-compliant laboratories. The built-in protocols for IQ/OQ/PQ ensure control of the consistent operation. Standardized PDF reports, user control and audit trails allow for adequate documentation of usage (Figure 3). With the Via2-Cassette™, the variabilities normally introduced during pipetting, staining and counting are minimized, thereby increasing the counting robustness and giving a highly precise result.
Literature:
- Singh, N., et al., Circulating apoptotic endothelial cells and apoptotic endothelial microparticles independently predict the presence of cardiac allograft vasculopathy. Journal of the American College of Cardiology, 2012. 60(4): p. 324-331.
- Bournazou, I., et al., Apoptotic human cells inhibit migration of granulocytes via release of lactoferrin. The Journal of Clinical Investigation, 2009. 119(1): p. 20-32.
- Brough, H.A., et al., IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy. Journal of Allergy and Clinical Immunology, 2014. 134(6): p. 1329-1338.e10.
- Buggert, M., et al., T-bet and eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection. PLoS Pathogens, 2014. 10(7): p. e1004251.
- Sandström, E., et al., Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus ankara. The Journal of infectious diseases, 2008. 198(10): p. 1482-1490.
- Hartmann, S.B., et al., Investigating the role of surface materials and three dimensional architecture on in vitro differentiation of porcine monocyte-derived dendritic cells. PLoS ONE, 2016. 11(6): p. e0158503.
- Wilson, G.A., et al., Human-specific epigenetic variation in the immunological leukotriene B4 RECEPTOR (LTB4R/BLT1) implicated in common inflammatory diseases. Genome Medicine, 2014. 6(3): p. 19-19.
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