Optimized processing of growth factor mobilized peripheral blood CD34+ products by counterflow centrifugal elutriation

Abstract

Cell separation by counterflow centrifugal elutriation has been described for the preparation of monocytes for vaccine applications, but its use in other current good manufacturing practice (cGMP) operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cell products were collected from healthy donors and processed by elutriation using a commercial cell washing device. Fractions were collected for each product as per the manufacturer's instructions or using a modified protocol developed in our laboratory. Each fraction was analyzed for cell count, viability, and blood cell differential. Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions. With modification of the basic protocol, we were able to collect all of the CD34+ cells in a single fraction. The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34, washed using the Elutra device, and transferred directly to a magnetic bead selection device for further purification. CD34+ cell purities from the column were extremely high (98.7 ± 0.9%), and yields were typical for the device (55.7 ± 12.3%). The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

Figure 1.

Elutriation process flowchart. Human granulocyte-colony stimulating factor-mobilized apheresis product is split into two products before elutriation if the number of WBCs is greater than 3 × 10¹⁰ or if the total volume of RBCs is greater than 7.5 ml (for protocol 1) or greater than 15 ml (for protocol 2). Abbreviations: HPC-A, hematopoietic progenitor cell apheresis; VRBC, volume of red blood cells; WBC, white blood cell.

Figure 2.

Fractionation of platelets, RBCs, and WBCs. (A): Profile of platelets, RBCs, and WBCs for protocol 1 for eight tissues. (B): Profile of platelets, RBCs, and WBCs for protocol 2 for two tissues (three elutriation runs). Abbreviations: RBC, red blood cell; WBC, white blood cell.

Figure 3.

Representative immunotype of elutriation fractions 2, 3, 4, and 5 of protocol 1 and fractions 1, 2, and 3 of protocol 2. (A): Flow cytometry plots of fractions 2, 3, 4, and 5 depicting FS versus SS, CD56 versus CD3, CD14 versus CD15, and CD34 versus CD19. (B): Flow cytometry plots of fractions 1, 2, and 3 depicting SS versus CD34. Abbreviations: FS, forward scatter; SS, side scatter.

Figure 4.

Comparison of RBCs and platelets after bag wash versus elutriated wash. Total red blood cell counts (n = 5) (A) and platelet counts (n = 4) (B) in samples in bag-washed and elutriated hematopoietic progenitor cell apheresis samples. Significance: p < .01 and p < .001, as indicated. Abbreviation: RBC, red blood cell.

Figure 5.

CD34 selection results. (A):CD34+ purity following bag centrifugation (n = 12) versus Elutra processing (n = 13). (B): Same as (A) for CD34+ recovery.

Figure 6.

Hematopoietic stem and progenitor cells (HSPCs) engrafted on NOD.Cg-prkdc^scid IL2rg^tm1Wjl/SzJ mouse. (A): Example of human cell engraftment in the bone marrow of a mouse 8.5 weeks after transplantation with HSPCs. (B): Analysis of spleen of same mouse. All subsequent analysis of lineage distribution is among the CD45+ population. Antibodies identify specific human cell lineages, as described in Materials and Methods. Abbreviation: SS, side scatter.