Manufacturing of cellular products for therapeutic purposes like stem cell or cancer therapy requires equipment with specific characteristics not always addressed by conventional technologies. A new integrated cell processing device is presented that can handle all current technical requirements for manufacturing cellular products by automation of the complete process in a GMP‐compliant single‐use tubing set. Its capabilities are exemplified in the presented study by successful processing of adult stem cells, natural killer cells, and several cell lines. Multiple cell processing workflows can be automated in a functionally closed environment: from cell separation through cell culture to formulation of the final product.

The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

BackgroundAutomation of cell therapy manufacturing promises higher productivity of cell factories, more economical use of highly-trained (and costly) manufacturing staff, facilitation of processes requiring manufacturing steps at inconvenient hours, improved consistency of processing steps and other benefits. One of the most broadly disseminated engineered cell therapy products is immunomagnetically selected CD34+ hematopoietic “stem” cells (HSCs).MethodsAs the clinical GMP-compliant automat CliniMACS Prodigy is being programmed to perform ever more complex sequential manufacturing steps, we developed a CD34+ selection module for comparison with the standard semi-automatic CD34 “normal scale” selection process on CliniMACS Plus, applicable for 600 × 106 target cells out of 60 × 109 total cells. Three split-validation processings with healthy donor G-CSF-mobilized apheresis products were performed; feasibility, time consumption and product quality were assessed.ResultsAll processes proceeded uneventfully. Prodigy runs took about 1 h longer than CliniMACS Plus runs, albeit with markedly less hands-on operator time and therefore also suitable for less experienced operators. Recovery of target cells was the same for both technologies. Although impurities, specifically T- and B-cells, were 5 ± 1.6-fold and 4 ± 0.4-fold higher in the Prodigy products (p = ns and p = 0.013 for T and B cell depletion, respectively), T cell contents per kg of a virtual recipient receiving 4 × 106 CD34+ cells/kg was below 10 × 103/kg even in the worst Prodigy product and thus more than fivefold below the specification of CD34+ selected mismatched-donor stem cell products. The products’ theoretical clinical usability is thus confirmed.ConclusionsThis split validation exercise of a relatively short and simple process exemplifies the potential of automatic cell manufacturing. Automation will further gain in attractiveness when applied to more complex processes, requiring frequent interventions or handling at unfavourable working hours, such as re-targeting of T-cells.

than targeting either alone. Erythropoietin-producing hepatocellular carcinoma-A2 (EphA2)-specific CAR T cells were used to target the A549 tumor cells. EphA2-specific T cells when administered together with FAP-specific T cells, resulted in a significant decrease in tumor growth and increased survival compared to mice that received either EphA2-or FAPspecific T cells alone. Our study underscores the value of cotargeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.

1188 An increasing number of clinical trials are enrolling patients in studies designed to examine the safety and efficacy of autologous stem cells for cardiac repair. Recent reports suggest that patients receiving CD133+ bone marrow cells after myocardial infarction, or as a treatment for ischemic cardiomyopathy, may benefit from an increase in global left ventricular function. Today the clinical scale enrichment of CD133+ cells has to be performed as a complex procedure involving numerous manual handling steps as well as a semi-automated magnetic separation process. We have developed a fully automated clinical scale process to purify CD133+ cells out of human bone marrow aspirates. The whole process was performed in a closed system, containing appropriate adaptors and tubing material, suitable for sterile connection of the bone marrow sample and required solutions, respectively. For the whole separation process, the total processing time was reduced from about 4.5 h (previous process) to 2.5 h. In this context, erythrocyte reduction, generation of autologous plasma, labeling time as well as the conditions for immunomagnetic separation of the CD133+ cells and the automatic monitoring of the whole process by a newly developed camera were optimized. To determine the reproducibility and stability of the process, CD133+ cells were separated from bone marrow aspirates with an initial volume of about 60 mL (n=10). The intitial frequency of CD133+ cells amounted to 0.34% (range: 0.11% to 0.66%) and the number of isolated CD133+ cells was 7.9×105 (range: 3.7×105 to 1.9×106). The yield was 47% (range: 23.9% to 50.9%) and the average viability of the separated CD133+ cells achieved 90% (range: 69.9% to 96.9%). The separation process typically achieved a >3.0 log depletion of CD133 negative cells, i.e. 99.9% of CD133 negative cells were removed. The log depletion of different cell types were: 4.0 for CD3+ cells (i.e. 99.99% removal), 3.1 for CD19+ cells, 3.4 for CD56+ cells, 3.2 for CD14+ cells, and 3.7 for CD15+ cells (n=3, respectively). After separation the CD133+ cells were automatically resuspended in 6 mL of clinical grade isotonic NaCl solution. For storage or transport of the cells, the NaCl solution could be automatically supplemented with 10% autologous plasma, generated out of the bone marrow sample during the separation process. The described cell separation system provides a safe and easy way to purify CD133+ cells from bone marrow aspirates within 2.5 h without any intermediate manual steps. The cell preparation in a closed sterile system facilitates a fast and robust enrichment of CD133+ cells. After separation the CD133+ cells are available in small volume and can be formulated for further use e.g. according to requirements for use in regenerative medicine. Disclosures: Essl: Miltenyi Biotec GmbH: Employment. Stuth:Miltenyi Biotec GmbH: Employment. Huppert:Miltenyi Biotec GmbH: Employment. Balshüsemann:Miltenyi Biotec GmbH: Employment. Bauer:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Employment.