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www.biopharminternational.com Quality and Regulatory Sourcebook eBook March 2022 BioPharm International 5 T cell into a CAR-T cell. Finally, the technician amplifies the CAR-T cells in a bioreactor and delivers the batch to the clinic, where a physician transfuses the cells back into the patient. Each step in this process depends on nature: technicians rely on viruses and T cell machinery to generate CAR-T cells as well as on the proper environ- mental conditions to expand the cells, while physicians rely on the patients' bodies to tolerate the cells and degrade them at an appropriate rate. Because developers and physicians cannot fully control all of these factors, they must monitor the creation and administra- tion of CAR-T cells to ensure they per- form as expected and will not induce adverse reactions in patients. Below are several sources of variability in CAR-T development and how ddPCR technol- ogy can help resolve some of the uncer- tainty inherent in the process. MEASURING CAR COPY NUMBER In the development of CAR-T cells, one key initial step is using viruses, such as adeno-associated virus (AAV) or lentivi- rus, in vitro to transfect T cells with the CAR gene. But once the initial condi- tions are set, it is difficult to control the behavior of these viruses and the genetic sequences they contain. As a result, the number of CAR transgenes that enter each cell and integrate into the genome can vary. Depending on the success of this transfection step, the treatment may not work at all, or worse, it may elicit a systemic inflammatory response that can cause further harm to patients (4). To protect patients, FDA recommends that CAR-T cell manufacturers performing viral transduction screen out cells con- taining less than one or more than four transgene copies per cell (5). To do this screening process, technicians need to quantify the CAR transgene copy num- ber accurately and precisely. Quantitative polymerase chain reac- tion (qPCR) is the standard method for quantifying nucleic acids, but it is not sensitive enough to quantify CAR transgenes with the accuracy and pre- cision needed to satisfy regulators. The qPCR technique quantifies nucleic acids indirectly, using a standard curve to estimate copy number. Scientists must generate this standard curve using serial dilutions, a process that is time-consum- ing and prone to human error. This error creates variability that makes it chal- lenging to quantify small copy numbers. If developers cannot tell with certainty whether or not a CAR-T cell batch con- tains at least one transgene copy per cell, they cannot be sure whether their trans- fection method was successful. ddPCR technology, however, can offer precise measurements of transgene copy number because of its ability to quantify gene sequences directly without using a standard curve. The technique involves partitioning a sample into approximately 20,000 nanoliter-sized droplets that con- tain one or a few nucleic acid strands each, with some droplets containing the target gene and some not. A separate PCR reaction takes place in each one. Starting with a sample of nucleic acids from CAR-T cells and using primers targeted to the CAR transgene, DNA amplif ication will only occur in the droplets containing that gene. As the gene amplifies, a reporter gets cleaved from the probe and emits a fluorescent signal. The f luorescent droplets can be counted to derive the transgene concen- tration in the original sample. Unlike qPCR, ddPCR does not measure the degree of amplification to estimate copy number in the original sample; instead, it counts whether or not amplification took place, providing a digital measure of copy number. This ability makes ddPCR technology more accurate and precise and, therefore, more reliable for assessing CAR copy number in CAR-T cells. These attributes were quantified in a study performed by Y. Luo et al. at the Huazhong University of Science and Technology in Wuhan, China (6). The researchers directly compared qPCR and ddPCR instruments in their ability to quantify DNA standards and CAR transgene copies in clinical peripheral blood samples from patients on CAR-T therapy. They found that ddPCR demonstrated greater sensitivity than qPCR. Specifically, among a series of DNA standards, ddPCR assays detected as few as 3.2 copies/mL, while qPCR showed a negative result at that con- centration. Furthermore, their ddPCR assay could reliably detect as few as five copies per reaction among clinical blood samples, while qPCR could not detect concentrations lower than 20 copies per reaction. Additionally, ddPCR tech- nology showed lower intra-assay and inter-assay coefficients of variance for the series of diluted standards than qPCR, suggesting that ddPCR is more repeat- able and reproducible. Mea nwh i le, resea rc hers at t he National Institutes of Health Clinical Center wanted to see if this method of copy number quantification was robust. To do this, P. Jin et al. tested the con- sistency of ddPCR assay results follow- ing several changes to the development workflow (7). Specifically, the research- ers transfected T cells using either len- tiviral or retroviral vectors. They then assessed transfection success right away or after the cells had been frozen for three or six weeks, using three different technicians across two different labora- tories. When they quantified CAR copy Quality and Regulatory Sourcebook Quality: Analytics Analytical tools such as droplet digital PCR can support CAR-T cell manufacturers in developing successful CAR-T cell products.