AAV vector production in suspension cells using PEI transfection and sodium butyrate with orthogonal assessment of function and quality.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, yet academic in-house production remains dominated by labor-intensive adherent cell workflows with limited scalability. Here, we describe an AAV vector production platform using suspension cells in orbital shaking Erlenmeyer flasks, based on polyethyleneimine (PEI) transfection and sodium butyrate supplementation. Following systematic evaluation of transfection conditions, this approach yields vectors with performance comparable to a commercial production kit. Vector quality was interrogated using orthogonal methodologies, including two-dimensional ddPCR, mass photometry, and nanopore sequencing, enabling comparative assessment of genome packaging, capsid composition, and vector heterogeneity. Functional validation was performed by in vitro transduction of K562 cells and primary human CD34+ hematopoietic stem and progenitor cells, as well as in vivo gene delivery to mouse liver and heart. Across assays, vectors produced using this protocol demonstrated comparable genome integrity and transgene expression. Comparative purification analysis revealed that iodixanol density gradient purification resulted in higher proportions of full capsids and reduced producer-cell-derived impurities relative to PEG 8000 precipitation. Together, this work establishes a scalable suspension-based AAV production workflow and demonstrates the value of orthogonal quality assessment combined with functional validation for robust vector benchmarking.