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

Abstract

Background: 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 20 639 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.

Methods: 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.

Results: 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 16 years) 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%.

Conclusion: 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.

Keywords: HLA; blood component production; blood donation testing; expansion; hematopoietic stem cell; quality control.