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6 BioPharm International Quality and Regulatory Sourcebook eBook March 2022 www.biopharminternational.com number amidst all of these protocol vari- ations, their ddPCR assay delivered con- sistent results. ddPCR technology was also central to optimizing P. Jin et al.'s transduc- tion protocol. The researchers varied the viral vector-to-T cell ratio as well as varied their centrifugation protocol (i.e., either centrifuging their samples or not) following transduction to iden- tify the best protocol for maximizing transduction efficiency and copy num- ber per cell. They used f low cytom- etr y to measure the effect of these variables on transfection efficiency while using ddPCR technology to measure copy number. Both of these measures were correlated throughout the exper- iments. Transfection eff iciency and copy number per cell increased as the vector-to-target cell ratio increased, but only when the samples were not cen- trifuged. Conversely, centrifuging the samples increased transfection efficiency at lower vector-to-target ratios. Overall, these data demonstrated that ddPCR technology can reliably quantify CAR transgene copy numbers across different conditions and can be used to assess the impact of various protocol deviations on the success of CAR-T cell production. DETECTING REPLICATION- COMPETENT VIRUSES A T cell with too many copies of the CAR gene can lead to immune reac- tions, but this scenario is not the only case in which a CAR-T cell could the- oretically pose a risk to patients. For example, CAR-T cell production using lentivirus could potentially create rep- lication-competent lentiviruses (RCL) that could infect patients and induce the rapid growth of T-cell neoplasms. This scenario is purely theoretical; however, to be safe, FDA recommends that manu- facturers test their clinical vector lots and manufactured cell products for RCL. In addition, the agency recommends that doctors test their patients' blood after receiving CAR-T cell therapy (8). ddPCR technology can be used to detect RCL. In one study, researchers T. Wiltshire et al. at the Mayo Clinic in Rochester, Minn., developed a ddPCR assay that detects the envelope sequence Quality and Regulatory Sourcebook Quality: Analytics CALL FOR PAPERS BioPharm International accepts four types of peer-review papers that are considered: standard data-driven, novel research; topical literature or patent review; technical case studies/technical application notes; and science-based opinion papers. Manuscripts for peer-review papers are accepted on an ongoing basis. Publication priority is given to papers in the order they are accepted for publication. Submitted papers are initially screened by the editors, then submitted for formal review by a member of the editorial advisory board, who will review the article for technical interest and content in a double-blind review process. Article acceptance is conditioned on the reviewer's approval. Once accepted for publication, a paper typically is published within three to five months. Peer-review papers are published in the print and digital editions of BioPharm International, and on www.BioPharmInternational.com. Links to the online versions of peer-review papers also are featured in e-newsletters distributed to the publication's audience. To learn more about the peer-review submission process, click the Submission Guidelines link on www.BioPharmInternational.com. C A L L FOR PAP ERS C A L L F O R P A P E R S CALL FOR PAPERS In the development of CAR-T cells, one key initial step is using viruses, such AAV or lentivirus, in vitro to transfect T cells with the CAR gene.