Clinimacs plus instrument

CD3⁺ cells were purified from healthy donors’ Leukopaks received from Hemacare (Northridge, CA, USA). Upon reception, the leukapheresis material was analyzed using the Sysmex pocH-100i Automated Hematology Analyzer (Sysmex, Milton Keynes, UK), diluted with CliniMACS PBS/EDTA buffer (Miltenyi, Bisley, UK), supplemented with 0.5% human AB serum (Seralab, Hayward Heath, UK) and centrifuged at 300 × g for 15 min at room temperature to remove platelets. The CD3⁺ fraction was purified from the plasma on the CliniMACS Plus instrument (Miltenyi) using the CliniMACS CD4 and CD8 reagents (Miltenyi) anti-CD4 and anti-CD8 monoclonal antibodies conjugated to superparamagnetic iron dextran particles. The viability of the final CD3⁺ product was assessed using the Vi-CELL XR (Beckman Coulter, High Wycombe, UK) and Nucleocounter NC-200 (ChemoMetec, Allerod, Denmark). The number of CD45⁺ cells, CD3⁺ cells, CD4⁺ cells, and CD8⁺ cells was evaluated before and after the cell separation on the CliniMACS by flow cytometry on the Fortessa (BD Biosciences, San Jose, CA, USA). The acceptance criteria for the CD3⁺ cell purification were set as follows: (1) viability over 95% and (2) number of CD3⁺ cells over 95%. Following purification, a working cell bank of CD3⁺ cells was cryopreserved in CryoStor CS10 (Sigma, Gillingham, UK) until use.

Leuokopaks were purchased from AllCells LLC and these were collected from healthy donors per standard protocols using mobilization with G-CSF+ Plerixafor. CD34+ cells were isolated from the leukopaks within 24 h by first removing the platelets using the LOVO cell Processing System (Fresenius Kabi). GMP grade reagents, buffers and columns for CD34 immunomagnetic selection were purchased from Milteny Biotec and the platelet washed cells were incubated for 30–35 min using the CD34 Reagent following which a subsequent wash for excess antibodies was performed on the LOVO. The washed and labelled cells were subject to immunomagnetic selection using the CliniMACS Plus instrument (Miltenyi Biotec) following which the cells were cryopreserved at a concentration of 5 × 10⁶ - 1 × 10⁷ cells/mL in CryoMACS 50 or 250 Bags (Miltenyi Biotec) for the gene edited drug product generation step.