GMP-grade CD34+ selection from HLA-homozygous licensed cord blood units and short-term expansion under European ATMP regulations.

Abstract

Based on a synergistic consortium, the cord blood (CB) bank Düsseldorf was responsible for the selection of HLA-homozygous (HLA-h) donors, contacting/re-consenting the mothers, Good Manufacturing Practice (GMP)-grade CD34+ enrichment, followed by short-term expansion of CD34+ cells and qualification of the resulting CD34+ population as advanced therapy medicinal product (ATMP)-starting material. Among 20639 licensed Düsseldorf cord blood units (CBUs), 139 potential HLA-h donors were identified with the most frequent 10 German haplotypes. 100% of the donors were contacted, and for 47·5%, consent was obtained. HLA-A, -B, -C, -DR, -DQ and -DP were determined by sequencing. Thawing/washing of the CBUs was performed in the presence of Volulyte/HSA with Sepax® , CD34+ selection by automated CliniMACS® -system (Miltenyi), expansion with qualified GMP-grade cytokines and media in the GMP facility. Here, we specify minimal criteria (≥5 x 105 viable CD34+ -count, ≥80% CD34+ -purity and ≥70% viability) and confirm that n=10 CB units (max storage time 16years) could be qualified for an ATMP starting material. The mean fold change expansion of isolated CD34+ cells at Day 3/4 (d3/4) was 3·38±3·02 with a mean purity of 86·90±10·38% and a high viability of 96·07±4·72%. As of March 2019, approval was obtained by the Bezirksregierung Düsseldorf for the GMP-compliant production. The production of HLA-homozygous expanded CD34+ cells from cryopreserved CB under European ATMP regulations presented here describes the successful clinical translation and implementation of a qualified manufacturing process. This approach considers the main obstacle of rejection of transplanted cells (due to the immunological HLA barrier) by preselection of HLA-homozygous transplants.