Charge‐Governed Mechanistic Evaluation of AAV2 Clarification via Functionalized Flat Sheet and Hollow Fiber Deconstructed Depth Filters

The expansion of adeno-associated virus (AAV)-based therapeutics has increased the demand for efficient clarification. In this study, a functionalized deconstructed depth filtration platform was developed to dissect how membrane-virus charge, modulated by pH, influences separation. Three charge regimes, positive (pH 4), neutral (pH 6), and negative (pH 8) between the membranes and AAVs, were examined. The flat-sheet system, composed of sequential 1.2, 0.8, 0.45, and 0.1 µm membranes, enabled the first layer-by-layer analysis of depth filtration behavior. This revealed that Stage 1 and Stage 4 dominate clarification. Remarkably, substantial DNA capture (> 75%) was observed even in the largest-pore stage, attributed to fouling layer formation that created a finer filtration barrier. Charge regime-dependent trends were evident: negative (pH 8) conditions produced the highest AAV2 recovery (> 90%) via electrostatic repulsion, while positive (pH 4) conditions yielded maximal impurity removal (> 85% DNA, > 70% protein reduction) through charge attraction. An optimized two-stage hollow fiber deconstructed depth filter (Stage 1: negative-negative; Stage 2: positive-negative) achieved balanced performance, attaining high AAV recovery and selectivity. This study establishes an analysis linking charge-governed fouling and selectivity to viral vector clarification performance, offering a rational framework for designing charge-engineered clarification systems for advanced gene therapy manufacturing.