Clinical manufacturing of CAR-T cells for adoptive immunotherapy
– using the NucleoCounter® instruments
The NucleoCounter® NC-200™ provides robust and accurate determination of cell count and viability of T cells during adoptive CAR-T cell manufacturing. The unique Via1-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 and DAPI, which are already pre-loaded into the Via1-cassette™. As conclusion, 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-200™
Figure 1: Monitoring the whole process from leukapheresis to the formulated product. With the NucleoCounter® NC-200™ 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 Via1-Cassette™ is pre-loaded the two dyes DAPI and Acridine Orange, which avoid some of the problems that lead to huge inaccuracies when bright field based counting is used. When counting samples containing erythrocytes, pre-treatment of the sample with an erythrocytes lysing buffer avoids the quenching effect of hemoglobin. Afterwards, the total cell count and the dead cell count are respectively determined by cell staining with Acridine Orange and DAPI using the Via1-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-200™. In conclusion, the NucleoCounter® NC-200™ 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 NucleoViewTM 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-200™ 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-200™ can be easily implemented in GMP and 21 CFR Part 11 compliant laboratories. The in-built 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 Via1-Cassette™, the variabilities introduced during pipetting, staining and counting are minimized, thereby increasing the counting robustness and giving a highly precise result.
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