In:
Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 2847-2847
Abstract:
CAR-T autologous cell therapies are delivering impressive results in the clinic. However, there are still significant manufacturing challenges impeding the rapid adoption of these advanced therapies. On the first day of cell processing, most manufacturing approaches require ~5 steps (~4 hours) to obtain a white blood cell (WBC) preparation sufficiently depleted of red blood cells (RBCs) for T-cell selection and activation steps; and involves large cell losses and a great deal of inconsistency. Here we present a single-step procedure that yields & gt;2 fold more cells that centrifugal processing with comparable or better quality in & lt;1 hour. We previously reported a small-scale microfluidic approach using deterministic cell separation (DCS) to effectively isolate and separate WBCs with high recoveries, no loss of WBC subtypes, no cell damage, and greater numbers of central memory T cells than traditional Ficoll-based processing. Extending this work, we now present the results of our fully scaled-up processing of 23 normal donor leukopaks and 4 disease samples using a full-scale DCS prototype. All samples were processed in & lt;45 minutes, with only an additional 10 minutes hands-on time. On average, inclusive of aggregate removal by prefiltering, DCS achieved 88% WBC recovery, 94% RBC removal, and 98% platelet ( PLT) removal from the undiluted leukopak samples (n=23). Furthermore, DCS resulted in a RBC/WBC ratio of 0.1 compared with a ratio of 1.4 for Ficoll. Similarly, the PLT/WBC ratios were 0.89 versus 7.17 for DCS and Ficoll, respectively (n=20). In addition, DCS preparations contained 2-fold more CD3+ T cells (n=17), and, importantly, the CD4+ cells were less differentiated (more cells in naïve and central memory stages) than those recovered by Ficoll. Similarly, DCS processed blood from cancer patients had a ratio of RBC/WBC = 7.0 versus 20.1 for Ficoll, and a PLT/WBC ratio = 0.7 versus 15.6 for Ficoll (n=4). These results demonstrate the capabilities of DCS in processing not only samples from normal donors but also blood from cancer patients with similar efficiencies. Further, with DCS we achieved wash efficiencies of more than 3 log, without the typically associated cell loss, as demonstrated by the removal of viral particles, soluble proteins and cytokines and growth factors present in plasma. Therefore, cells from leukopaks processed by DCS can be washed and collected directly into cell culture media, or other solutions, to ready them for downstream applications without pelleting and repeated washes, greatly simplifying workflows. We integrated our DCS technology into a full scale parallelized, disposable, closed fluid path solution and automated platform prototype, the Curate ® Cell Processing System, capable of processing undiluted leukopacks at rates in excess of 300mL/hour. Designed to process blood products in bags using a single-use cassette containing microfluidic components, the Curate ® delivers a debulked WBC product to a bag. With a hands-on time of only 10 minutes, the Curate ® reduces the time to activation- and expansion-ready cells from leukopaks by 6-fold as compared with centrifugation and elutriation methods (Bowles, et al. Cytotherapy 2018;20(5):S109). The system can process a full leukopak (200-300 mL containing up to 1.2x10 10 WBC) within 40 minutes with a maximal cell throughput of 1.8x10 10 WBC per hour. Additionally, the same Curate ® device can be used to achieve up to 200x10 6 cell/mL in as little as 40 mL of media and without requiring pelleting. In summary, we believe our technology enables a significant breakthrough in the production of CAR-T cells by efficiently recovering more and cleaner total and naÏve T cells, for CAR-T cell production. Furthermore, the closed-system Curate ® will simplify cell processing workflows by reducing the number of cell washing steps, as well as the hands-on time and resources. Supported in part by NIH Grant No 5R42CA228616-03 Disclosures Behmardi: GPB Scientific, Inc: Current Employment. Ouaguia: GPB Scientific, Inc: Current Employment. Healey: GPB Scientific, Inc: Current Employment. Jones: GPB Scientific, Inc: Current Employment. Rahmo: GPB Scientific, Inc: Current Employment. Skelley: GPB Scientific, Inc: Current Employment. Gandhi: GPB Scientific, Inc: Current Employment. Campos-Gonzalez: GPB Scientific, Inc: Current Employment, Current holder of stock options in a privately-held company. Civin: GPB Scientific, Inc: Current holder of individual stocks in a privately-held company. Ward: GPB Scientific, Inc: Current Employment.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood-2021-153528
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2021
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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