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The rapid development of adeno-associated viral vectors (AAV) to treat genetic disease has placed increased emphasis on the design of efficient downstream manufacturing processes. This study investigated the potential of using single pass tangential flow filtration (SPTFF) as a novel means of concentrating and purifying AAV clarified cell lysate (CCL). AAV stability studies revealed the shear-sensitive nature of the AAV capsids, with evidence of aggregation and fragmentation following repeated passages through a peristaltic pump (as would occur during batch ultrafiltration). SPTFF experiments focused on first identifying the membrane(s) that permitted high yield of AAV (negligible sieving into the permeate) along with substantial host cell protein (HCP) removal. Experiments were then performed at various permeate fluxes, which revealed that stable SPTFF processes can be achieved by operating below a critical flux for fouling (Jfoul). 300 kDa regenerated cellulose (RC) membranes were identified as optimal for this application, given their ability to provide complete AAV retention with high removal of HCP (>90%) when operated below Jfoul. The critical flux during SPTFF was increased by preconditioning the CCL through a positively-charged adsorptive filter, which reduced the concentration of foulants prior to SPTFF. These studies provide the first demonstration of SPTFF for the concentration and purification of AAV clarified cell lysate while minimizing shear exposure. 

ABSTRACT To enable adeno‐associated viral vectors (AAV) to achieve their maximum potential, next‐generation manufacturing processes must be developed to make gene therapies more affordable and accessible. This study focused on the design of two different intensified AAV downstream manufacturing processes at bench and pilot scale. Novel clarification methods were studied at bench scale, including the use of BioOptimal™ MF‐SL tangential flow microfilters for continuous removal of cell debris. Membrane adsorbers were used for further clarification, including DNA removal. Single pass tangential flow filtration (SPTFF) was implemented at bench scale by feeding the clarified cell lysate (CCL) into two Pellicon XL50 cassettes with 100 kDa regenerated cellulose membranes. At pilot scale, a multi‐membrane staged SPTFF module was designed to concentrate 10 L of AAV CCL. Both SPTFF systems provided 12X inline volumetric concentration with AAV yield > 99% after an appropriate buffer chase. Host cell protein removal was 48% and 37% for the bench and pilot scale processes, respectively. As an initial proof‐of‐concept, an integrated process was developed at pilot‐scale which linked clarification, SPTFF, and affinity chromatography. The integrated process offered an 81% reduction in total operating time (due to the reduced volume of load material for the affinity column after preconcentration by SPTFF), 36% improvement in affinity resin utilization (due to the higher AAV concentration in the column load), and an estimated 10% reduction in raw material costs. These improvements translated to an 8.5‐fold increase in overall productivity compared to an equivalent batch process, underscoring the potential for SPTFF to intensify large‐scale AAV downstream processing. 

The influence of salts on the solvophobic interactions of two non-polar surfaces in organic solvent was examined using a series of molecular balances. Specific anion effects were observed that followed the Hofmeister series and enhanced the solvophobic effect up to two-fold.