Clinimacs instrument

A two-step cell processing procedure that involved selection of CD34⁺ cells followed by depletion of CD45RA⁺ cells was performed on G-PBSC apheresis products using the CliniMACs instrument (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). CD34⁺ selections were performed using the CliniMACS CD34 reagent system (20 (link), 39 (link)). A GMP-grade murine αCD45RA mAb (clone T6D11) directly conjugated to Miltenyi iron dextran beads was produced by Miltenyi Biotec under contract from the NIH RAID (Rapid Access to Intervention Development) program and used for depletion of CD45RA⁺ cells from the CD34⁻ fraction. In brief, processing involved the following steps: G-PBSC apheresis products were obtained from donors, sampled and stored overnight at 4°C prior to processing. If the cell concentration of the product was greater than 200 × 10⁶ cells/mL, the volume was increased by the addition of 1% human serum albumin (HSA)/Normosol-R to reduce the cell concentration to ≤ 200 x10⁶ cells/ml. The total nucleated cell and CD34⁺ cell numbers were used to determine whether to proceed to cell processing immediately after overnight storage or to pool the initial apheresis product with a second product collected the following day.

Specifications for method(s) of apheresis collection of G-CSF mobilized peripheral blood stem cells (PBSCs) were per site-specific practice and were not regulated by our DMF. We defined the start of the manufacturing process for Auto-CD34⁺HPC at the cell processing facility upon receipt of the apheresis product, which was considered the raw material. Process flow per the Master Production Batch Record (Supplementary Appendix S2, SOP 3001) provided for purification and characterization before cryopreservation and storage.
For CD34⁺ enrichment, we used the Baxter Isolex (Baxter Healthcare Corporation, One Baxter Parkway, Deerfield, IL) 300i Magnetic Cell Selection System per the manufacturer’s instructions, which specified a device load of ≤1000 mL containing ≤8 × 10¹⁰ TNC [7 ]. When production of Isolex was discontinued in 2010, we amended our DMF and the SCOT protocol, which had not yet completed enrollment, for substitution with the Miltenyi CliniMACS Instrument. Additional process flow specifications were per NIAID SOPs.
We defined a manufacturing “lot” as the product from 1 run of Isolex or CliniMACS CD34⁺ cell selection, containing ≥3.5 × 10⁶ viable cells. Depending on variables of the apheresis collection, processing of an individual subject’s graft required manufacture of 1 or more lots of Auto-CD34⁺HPC.