ABSTRACT 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.
Baughman‐Leach et al. (Wed,) studied this question.