Prospective Evaluation of a Practical Guideline for Managing Positive Sterility Test Results in Cell Therapy Products.

Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products that fail release criteria (because of potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies. We developed a systematic protocol for the assessment of positive microbial sterility test results using retrospective data from 2007 to 2016. This protocol was validated and applied prospectively between October 2016 and September 2017 to 13 products from which positive sterility results had been reported. Viable and nonviable environmental monitoring (EM) data were collected concurrently as part of a facility control assessment. Three of 13 (23%) positive sterility results were attributable to bone marrow collections that had been contaminated with skin flora during harvest; all were infused without pertinent infectious sequelae. Of the remaining 10, 1 was deemed a true positive and was discarded before infusion, whereas 9 were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and 6 were withheld because of patient issues unrelated to microbial sterility results. No postinfusion-associated infectious complications were documented. Almost half of the positive EM findings were skin flora. Paired detection of an organism in both product and associated EM was identified in 1 case. Application of our validated protocol to positive product sterility test results allowed for systematic data compilation for regulatory evaluation and provided comprehensive information to clinical investigators to ensure timely and strategic management for product recipients.

Effects of starting cellular material composition on chimeric antigen receptor T-cell expansion and characteristics.

BACKGROUND: When manufacturing chimeric antigen receptor (CAR) T cells using anti-CD3/anti-CD28 beads, ex vivo T-cell expansion is dependent on the composition of leukocytes used in the manufacturing process. We investigated the effects of leukocyte composition on CAR T-cell expansion and characteristics using an alternative manufacturing method. METHODS: Anti-B-cell maturation antigen and CD19-CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T-cell culture initiation. T-cell expansion was stimulated with soluble anti-CD3 and interleukin-2. RESULTS: Fifty-one CAR T-cell products were evaluated; 28 anti-B-cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T-cell expansion was reduced when greater quantities of monocytes were present in the post-density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T-cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post-density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells. CONCLUSIONS: The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T-cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T-cell products.

Robust Selections of Various Hematopoietic Cell Fractions on the CliniMACS Plus Instrument.

Cell separation technologies play a vital role in the graft engineering of hematopoietic cellular fractions, particularly with the rapid expansion of the field of cellular therapeutics. The CliniMACS Plus Instrument (Miltenyi Biotec) utilizes immunomagnetic techniques to isolate hematopoietic progenitor cells (HPCs), T cells, NK cells, and monocytes. These products are ultimately used for HPC transplantation and for the manufacture of adoptive immunotherapies. We evaluated the viable cell recovery and cell purity of selections and depletions performed on the CliniMACS Plus over a 10-year period at our facility, specifically assessing for the isolation of CD34+, CD4+, CD3+/CD56+, CD4+/CD8+, and CD25+ cells. Additionally, patient- and instrument-related factors affecting these parameters were examined. Viable cell recovery ranged from 32.3 ± 10.2% to 65.4 ± 15.4%, and was the highest for CD34+ selections. Cell purity ranged from 86.3 ± 7.2% to 99.0 ± 1.1%, and was the highest for CD4+ selections. Undesired cell fractions demonstrated a range of 1.2 ± 0.45 to 5.1 ± 0.4 log reductions. Red cell depletions averaged 2.12 ± 0.68 logs, while platelets were reduced by an average of 4.01 ± 1.57 logs. Donor characteristics did not impact viable cell recovery or cell purity for CD34+ or CD4+ cell enrichments; however, these were affected by manufacturing variables, including tubing size, bead quantity, and whether preselection platelet washes were performed. Our data demonstrate the efficient recovery of hematopoietic cellular fractions on the CliniMACS Plus that may be optimized by adjusting manufacturing variables.