Affinity Peptides With pH Sensitivity for the Enrichment of CD38⁺ Cells

The selective enrichment of cell populations based on surface markers is critical for the advancement of gene and cell therapies. Current antibody-based cell isolation methods, such as fluorescence- and magnetic-activated cell sorting (FACS and MACS), offer high specificity but are limited by scalability, cost, and potential adverse effects on cellular physiology, including differentiation or apoptosis. In this study, we present an alternative antibody-free approach for reversible cell isolation using pH-responsive peptides that target the CD38 surface marker. Through in silico design, we developed affinity peptides with pH Sensitivity (APPS) that selectively bind CD38 at physiological pH and release target cells under mildly basic conditions (pH 8). The peptides were conjugated to amine-functionalized magnetic beads at controlled surface densities (1.25-40 equivalents) and evaluated for their performance in isolating CD38⁺ hematopoietic cells from a mixed population of RPMI 8226 (CD38⁺) and K562 (CD38^-) cells. Compared to antibody-based MACS, APPS-functionalized beads achieved superior CD38⁺ cell purity (> 80% vs. > 50%) while maintaining high cell viability (~90%). The integration of APPS beads into a microfluidic platform enabled pseudo-continuous cell separation with elution rates exceeding 10⁵ cells·mL^-1·min^-1. These results demonstrate that APPS beads provide a gentle, scalable, and reversible alternative for cell isolation, with significant potential for analytical and preparative applications in cellular therapy manufacturing.