The purpose of the test is to provide rapid less than 15minutes quantitative Endotoxin results for in-process and point of use samples. Each lot of cartridges has a pre-calculated CSE reaction time curves already programmed into the system. Once the lot number of the cartridge being used is entered into the PTS reader the system will calculate the result based on the pre-programmed standard curve reaction times. There are two assay sensitivities available for use: The standard curve reaction times will be based on the sensitivity of the assay.
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Abstract Background Rapid release testing reduces the waiting period for administration of time-sensitive cell therapy products. Current assay systems are labor intensive and time consuming. Subsequently, each center tested endotoxin content of standard dilutions of cell therapy products thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing.
Endotoxin detection between paired results was equivalent in most cases. The toxin exerts its effects by inducing pyrogenicity and increasing cell wall permeability. Clinically, endotoxin can result in diarrhea, septic shock, complement activation, marrow necrosis, and multiple other effects [ 1 ].
Testing for endotoxin has, therefore, become a standard release test for cellular and gene therapy products that are manufactured using in vitro culture or manipulation, and for certain non-homologous products that are to be administered without cryopreservation.
The maximum acceptable level of endotoxin in these products is usually 5. When LAL was first introduced in the s it was limited to the gel clot method, which employed multiple dilutions of the sample and controls. Today, kinetic methods utilizing microtiter plate readers are more common. Partly because of its sensitivity to technical variables, the LAL assay is notoriously demanding.
Very careful set-up and pyrogen-free materials, including accessories and water, must be used. Creation of dilutions to construct standard curves and addition of control standard endotoxin spikes to test for possible inhibition or enhancement by the test sample, add another layer of complexity. These details influence the performance of the endotoxin assay.
Failure of the assay for technical reasons and consequent repetition can delay infusion of non-cryopreserved products for hours, potentially affecting product quality and patient well-being.
Methods Participants A total of five cell therapy-manufacturing laboratories at four facilities participated in this evaluation. Cartridges with 0. The cartridges are potency tested, spike recovery is performed, and the calibration code is determined. The calibration code contains the cartridge test parameters that were determined during potency testing, as well as the archived curve for that batch of cartridges.
The calibration code is then verified with an additional test cartridge. The cartridge utilizes kinetic chromogenic LAL formulations in a modified format, which measures color intensity proportional to endotoxin concentration.
Each cartridge consists of two sample channels and two spiked channels, consistent with current FDA guidance for licensed quantitative LAL methods. Each reservoir contains a specific amount of LAL reagent, synthetic chromogenic substrate, control standard endotoxin CSE , and buffers uniformly imbedded in the cartridge [ 4 ]. Formulation of new substrates with each run is, therefore, eliminated for the user.
Using an internal pump, the reader draws, mixes, and incubates the sample with the various reagents at programmed time intervals before transferring it to the optical chambers. The portable spectrophotometer then monitors the change in the optical density and calculates the endotoxin level based on the resulting kinetic values.
Samples need to have reaction times that can be plotted on the archived curve. Results greater than 5. With the correct dilution the unit achieves results in approximately 15 minutes [ 4 ].
Samples that are positive for endotoxin will react faster.
Charles River Endosafe-PTS Manuals