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

Abstract

A B S T R A C TCell 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.

Design and validation of a GMP stem cell manufacturing protocol for MPSII hematopoietic stem cell gene therapy

“… 26 We performed TD optimization studies using cryopreserved human CD34 + cells (hCD34), previously isolated from a healthy donor leukapheresis unit by magnetic bead separation using a CliniMACSplus instrument. 27 We compared a range of IDS.ApoEII LV concentrations (12.5, 25, 50, and 100 MOI) with and without the TEs LentiBOOST and protamine sulfate, as tested in similar TD optimization studies, albeit with different LVs. 28 , 29 The growth media chosen for these studies was serum-free X-VIVO-15 with the inclusion of 1% human albumin serum (HAS) and fms-related tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), thrombopoietin (TPO) and interleukin-3 (IL-3), as used in a number of GMP cell manufacturing protocols for ADA and CGD.…”

CD3/CD19 Depletion for T-cell Reduction of Allogeneic Transplants: Mostly Efficient, but not Robust

“…Thirdly, it is apparent that T-cell- and particularly B-cell-depletion by CD3/CD19-depletion is markedly less efficient than by CD34 selection, by approximately one and two common log. Depletion of T-cells in CD34 selected grafts was shown in several studies to lie in the range of −5 to −6 log depletions [ 22 , 33 , 34 ], whereas T-cell depletion in CD3/CD19 depleted grafts did not exceed −4 log depletion [ 22 ].…”