Background: Relapse and opportunistic viral infections remain major causes of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Adoptive transfer of natural killer (NK) cells is a promising strategy to enhance post-transplant immune reconstitution. Adaptive NKG2C+ NK cells exhibit enhanced cytotoxicity and persistence, but the influence of baseline donor immunophenotype on GMP manufacturing has not been systematically investigated. We evaluated whether donor NKG2A/NKG2C immunophenotypes are associated with successful manufacture of adaptive NK-cell products. Methods: Eighty-three healthy donors from the ReDoCel registry underwent immunophenotypic characterization of circulating NK-cell subsets by multiparametric flow cytometry. Donors were stratified by unsupervised hierarchical clustering according to NKG2A/NKG2C expression. Representative donors from NKG2C- and NKG2A-dominant clusters underwent feeder-free GMP-compliant expansion using the automated CliniMACS Prodigy® platform (Miltenyi Biotec, Bergisch Gladbach, Germany). Expanded products were evaluated for manufacturing efficiency, immunophenotype, cytotoxic function, and post-thaw stability. Results: Baseline NKG2C frequencies showed marked inter-donor variability, allowing identification of four immunophenotypic clusters. Only the NKG2C-dominant donor achieved successful GMP manufacturing, exceeding the predefined expansion threshold while maintaining high viability and purity. Both NKG2A-dominant donors showed limited proliferative capacity under identical manufacturing conditions. Expanded NK cells acquired an activated phenotype characterized by increased expression of DNAM-1, NKG2D, NKp30, NKp46, and TIM-3 while preserving mature differentiation and KIR expression. Functional analyses demonstrated potent degranulation against leukemia targets with minimal autoreactivity, resulting in a predominantly cytotoxic effector profile. The successfully expanded product maintained viability, phenotype, and function after long-term cryopreservation. Conclusions: This proof-of-concept study suggests that baseline donor NKG2A/NKG2C immunophenotype may influence GMP manufacturing of adaptive NK-cell products. These findings support prospective evaluation of donor immunophenotyping as a biomarker for donor qualification and manufacturing optimization to facilitate standardized off-the-shelf adaptive NK-cell therapies after allo-HSCT.

Background/Objectives: Post-thaw washing of cryopreserved hematopoietic progenitor cell (HPC) products is performed to remove dimethyl sulfoxide (DMSO) and reduce infusion-related toxicity. Hydroxyethyl starch (HES) has traditionally been used as an osmotic buffer in washing solutions; however, severe regulatory restrictions and market suspensions by the European Medicines Agency (EMA) have limited its availability in Europe. This pilot study evaluated the technical feasibility of utilizing a 4.0% human serum albumin (HSA)-based washing solution to replace HES without compromising post-thaw product quality parameters. Methods: Sixteen cryopreserved peripheral blood HPC bags from eight patients (cryopreserved between 2006 and 2015) were analyzed using a paired experimental design. For each patient, one bag was thawed and washed using the standard HES-HSA solution, while the paired bag was processed using an HSA-based solution. Total nucleated cell (TNC) recovery, TNC viability, viable CD34+ cell recovery, CD34+ viability, and clonogenic potential (CFU assay) were assessed immediately after washing and after 90 minutes of short-term post-thaw storage. Results: No statistically significant differences were observed between the HES-HSA and HSA washing solutions for any evaluated parameter. TNC recovery rates were 86.6 ± 6.6% and 88.9 ± 7.9% for the HES-HSA and HSA groups, respectively (p = 0.54). Post-thaw viability, viable CD34+ cell recovery, CD34+ viability, and clonogenic potential were stable and comparable between both processing strategies. Conclusions: An albumin-based washing solution provides cellular recovery, viability, and clonogenic performance comparable to conventional HES-containing formulations in a laboratory setting. Given current restrictions on HES due to clinical safety concerns, an albumin-based formulation represents a practical and logistically viable operational alternative for the post-thaw processing of peripheral blood HPC products.