Endotoxin quality control testing for CAR T-cell manufacturing: validation considerations for Endosafe portable testing system

• Published in: Cytotherapy (Oxford, England) Vol. 22; no. 5; p. S140
• Main Authors: Barry, N., Velickovic, Z., Rasko, J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.05.2020
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2020.03.279

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. To determine endotoxin limits, maximum valid dilution of samples and reproducibility of the Endosafe-PTS test. 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. 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. 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.