Quality Control Testing

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William Michael Dunne - One of the best experts on this subject based on the ideXlab platform.

S. Nanua - One of the best experts on this subject based on the ideXlab platform.

John E.j. Rasko - One of the best experts on this subject based on the ideXlab platform.

  • endotoxin Quality Control Testing for car t cell manufacturing validation considerations for endosafe portable Testing system
    Cytotherapy, 2020
    Co-Authors: N Barry, Z. Velickovic, John E.j. Rasko
    Abstract:

    Background & Aim Background Adoptive cell therapies have emerged as promising treatments for cancer patients. The manufacture of clinical-grade Chimeric Antigen Receptor (CAR) T-cells therapies encompass multiple complex processes under the code of good manufacturing practice (cGMP) to ensure high-Quality and safe products. Endotoxins are lipo-polysaccharides from gram-negative bacteria that cause serious adverse events in patients. For this reason, it is important that CAR T-cells are tested for endotoxin content. The limulus amoebocyte lysate (LAL) test is FDA approved and allows sensitive detection of bacterial endotoxins. The Endosafe Portable Testing System (PTS) is a rapid, FDA approved Testing system manufactured by Charles River that uses disposable cartridges for accurate real-time endotoxin Testing, However, a number of important parameters need to be considered when establishing Endosafe-PTS Testing for CAR T-cells products. Aim To determine endotoxin limits, maximum valid dilution of samples and reproducibility of the Endosafe-PTS test. Methods, Results & Conclusion Methods Inhibition and enhancement screening cartridges were used initially to establish maximum valid dilution and investigate potential inhibition of the test. CAR T-cells were manufactured from three individuals and analysed for endotoxins on the Endosafe-PTS using cartridges with a sensitivity of 5-0.05 EU/mL and spike recovery 50-200% according to manufacturer's protocol. Three replicates of each sample were tested. Results The optimal dilution of CAR T-cell product was determined at 1:20 with the spike recovery of 84% to 130%. No inhibition was detected at the 1:20 dilution for all three samples. The upper limit of acceptance criteria for endotoxin was determined on the basis of dose and FDA guidelines to be 5 EU/kg body weight/hour of intended product administration. Conclusion The Endosafe-PTS provides an accurate, reproducible and rapid method for Endotoxin detection which is crucial in Quality Control release Testing and administration of time-sensitive CAR T-cell products. It is important to validate inhibition and enhancement screening for each CAR T-cell product and determine the optimal sample dilution before implementation of the Endosafe-PTS for product release Testing.

  • Vector copy number Quality Control Testing for CAR T-cells: critical validation parameters
    Cytotherapy, 2020
    Co-Authors: A. Chen, Z. Velickovic, John E.j. Rasko
    Abstract:

    Background & Aim Background Retroviral and lentiviral vectors have been broadly used in Chimeric Antigen Receptor (CAR) T-cell therapy clinical trials. These vectors have the capacity to integrate permanently into host cell DNA. There is an increased risk of oncogenesis if the vector copy number (VCN) per cell is high. The Food and Drug Administration (FDA) recommends that the VCN shall be Aims Establish a reliable test for detection of VCN in patient samples. Evaluate the linearity of the vector copy number qPCR and primer multiplexing. Methods, Results & Conclusion Methods CD3+ T-cells from three individuals with no tumour burden were transduced using a retroviral vector to generate CAR T-cells. CAR T-cells and the Un-transduced T-cells (Controls) were expanded in the same culturing conditions for 14 days. Genomic DNA was extracted from the six samples using Qiagen DNA extraction kit. Human albumin (huALB) and the retroviral vector backbone copy numbers were detected using qPCR with TaqMan probes against a standard curve. Results The Vector Copy Number of the CAR-T cell products were 5, 3, and 2 copies for Donor 1, 2 and 3, respectively. Multiplexing of huALB and vector probes did not impact the accuracy of VCN detection. The linear correlation coefficient was r2 = 0.992 ± 0.003. Conclusion qPCR is a sensitive and reliable Quality Control test for detection of VCN in CAR T-cells. A number of critical process parameters and key variables need to be considered when preparing VCN validation protocols.

L. Isgriggs - One of the best experts on this subject based on the ideXlab platform.

Carol J. Weber - One of the best experts on this subject based on the ideXlab platform.

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