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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. 

ABSTRACT Single Pass Tangential Flow Filtration (SPTFF) is increasingly used for inline concentration and final formulation in intensified/continuous processes for monoclonal antibody products. However, these modules typically operate at low feed flux, requiring significant membrane area and often complex internal staging to achieve the desired concentration factor. In this study, a vibration‐assisted SPTFF system was used for inline concentration of soluble protein. The maximum sustainable flux and concentration factor were evaluated under vibratory and non‐vibratory conditions using flux‐stepping experiments. SPTFF performed under vibration was able to achieve single pass concentration factors of 20× at a feed flux of 17.2 L/m²/h, while the non‐vibratory system showed rapid fouling at much lower concentration factors. Furthermore, the vibratory module achieved a 6‐fold higher concentration factor compared to a screened channel cassette. Long‐term filtration experiments demonstrated that the vibratory system could concentrate a 20 g/L protein solution to 100 g/L using a single cassette with stable operation for more than 8 h without protein aggregation. This work highlights the potential opportunity to develop vibratory SPTFF systems for intensified bioprocessing. 

Abstract The design of effective ultrafiltration/diafiltration processes for protein formulation requires the use of membranes with very high protein retention. The objective of this study was to examine the effects of specific buffers on the retention of a model protein (bovine serum albumin) during ultrafiltration. Albumin retention at pH 4.8 was significantly reduced in phosphate buffer compared with that in acetate, citrate, and histidine. This behavior was consistent with a small change in the effective albumin hydrodynamic diameter as determined by dynamic light scattering. The underlying conformational changes leading to this change in diameter were explored using circular dichroism spectroscopy and differential scanning calorimetry. These results provide important insights into the factors controlling protein retention during ultrafiltration and diafiltration. 

Protein fouling can significantly reduce the filtrate flux, capacity, and virus retention during processing of plasma- or mammalian cell-derived biopharmaceuticals through virus removal filters. We use focused ion beam (FIB) milling and scanning electron microscopy (SEM) to directly evaluate changes in 3D pore structure in a Viresolve® Pro membrane due to fouling by human serum immunoglobulin G. Protein fouling causes a significant reduction in the membrane porosity, which decreases by approximately 40% in the size-selective region near the exit of the highly asymmetric Viresolve® Pro membrane after the filter is fouled to 90% flux decline. There is a corresponding reduction in the number of small pores by more than a factor of two. Model simulations of flow and particle transport in the protein-fouled membrane are in good agreement with independent experimental measurements of the permeability and location of particle capture. Simulations show an upstream shift in the location of nanoparticle capture (away from the filter exit) by about 0.4 µm for the membrane fouled to 90% flux decline. This is due to pore constriction from protein deposition, highlighting how fouling redistributes flow paths within the membrane. These results demonstrate the capability of using FIB-SEM to directly evaluate the effects of protein fouling on the 3D pore structure in virus removal filters, providing important insights into how protein fouling alters the performance of these highly selective membranes. 

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. 

Forward osmosis (FO) has primarily been explored for applications in water desalination. While FO has also shown potential in concentrating dairy products, little to no attention has been paid to its potential in concentrating biotherapeutics, particularly to the very high concentrations needed for many monoclonal antibody products that are delivered by subcutaneous injection. This study demonstrates the feasibility of using FO as an alternative to ultrafiltration (UF) to achieve highly concentrated protein formulations using human Immunoglobin G (hIgG) as a model protein. The permeate flux in FO, using 1 M NaCl as the draw solution, decreased with increasing hIgG concentration due primarily to concentration polarization effects that are strongly influenced by the increase in feed viscosity for the concentrated hIgG solution. The importance of the hIgG viscosity on the FO performance was demonstrated by performing experiments with concentrated polyethylene glycol solutions and through mathematical modeling that accounts for the effects of both external and internal concentration polarization on FO performance. Batch concentration experiments with FO achieved final hIgG concentrations greater than 290 g/L compared to a maximum achievable concentration in UF of approximately 150 g/L. These results clearly demonstrate the potential of using FO, with high osmotic pressure draw solutions, to achieve highly concentrated formulations of therapeutic proteins that are beyond the capability of current UF processes. 

Recent advances in the use of viral vectors for gene therapy has created a need for efficient downstream processing of these novel therapeutics. Single-pass tangential flow filtration (SPTFF) can potentially improve final product quality via reductions in shear, and it can increase manufacturing productivity via simple implementation into continuous/intensified processes. This study investigated the impact of variations in pressure and flow rate along the length of the membrane on overall SPTFF performance. Constant-flux filtration experiments at feed fluxes from 14 to 420 L/m2/h (Reynolds numbers <20) were performed using Pellicon® 3 TFF cassettes with fluorescent nanoparticles as model viral vectors. The location of nanoparticle accumulation shifted towards the filter outlet at high conversion and was also a function of the permeate flow configuration. These phenomena were explained using a newly developed concentration polarization model that predicts the distribution in local wall concentration over the length of the membrane. The model accurately captured the observed nanoparticle accumulation trends, including the effects of the permeate flow profile (co-current, divergent, or convergent flow) on nanoparticle accumulation within the SPTFF module. Nanoparticle accumulation at moderate conversion was more uniform using convergent flow, but nanoparticle accumulation at 80 % conversion (5x concentration factor) can be minimized using a divergent flow configuration. The local wall concentration model was also used to evaluate the critical flux by assuming that fouling occurs when the nanoparticle concentration at any point along the membrane surface exceeds 15 % by volume. These results provide important insights for the design and operation of SPTFF technology for inline concentration of viral vectors. 

In the biopharmaceutical industry, virus filters are crucial for ensuring the removal of endogenous and adventitious viruses as part of the viral clearance strategy. Although traditionally described as a size-exclusion mechanism, virus retention has a pro-cess-dependent nature where challenging conditions, such as process disruptions, may compromise membrane retention and significantly increase virus filtrate concentrations. The detailed mechanisms underlying this loss of retention are challenging to determine using traditional breakthrough experiments. In this work, single particle tracking and kinetic simulations were employed to connect individual particle behavior to the observed macroscopic losses in virus retention. Our experiments, using fluorescently labeled ΦX174 bacteriophage as a model parvovirus, replicated conditions representative of process disruptions within the Pegasus SV4, a homogeneous polymeric virus filtration membrane. During flow, phage particles retained were trapped within relatively large cavity spaces that had downstream constrictions aligned with the flow direction; the trapped particles were dynamic and exhibited significant intra-cavity motion. Upon flow stoppage, particles escaped from these retention locations rapidly, with approximately 90% of previously trapped particles being remobilized for process dis-ruption time ranging from 2 to 10 minutes, suggesting that local cavity escape had reached saturation at these timescales. Diffusion experiments within the membrane revealed isotropic and Fickian motion, hindered by more than an order of mag-nitude compared to diffusion in unconfined liquid. Despite the reduced mobility within the membrane, the substantial diffusion coefficient of 4.19 ± 0.06 µm²/s indicated that virus particles could travel tortuous but non-retentive pathways through the membrane on length scales equal to or greater than the membrane thickness during a disruption event. A 1D kinetic Monte-Carlo simulation successfully connected single-particle behavior to macroscopically observed virus release, indicating that significant diffusive release into the filtrate can occur even without the resumption of flow. This work provides crucial insights into the retention behavior of homogeneous membranes during periods of disruption, enabling the design of more robust mitigation strategies and filter designs. 

The performance of virus filters is often determined by the extent of protein fouling, which can affect both filtrate flux and virus retention. However, the mechanisms governing changes in virus retention in the presence of proteins are still not well understood. The objective of this work was to examine the effect of proteins on virus retention by both asymmetric (Viresolve® NFP and Viresolve® Pro) and relatively homogeneous (Ultipor® DV20 and PegasusTM SV4) virus filtration membranes. Experiments were performed with bacteriophage ϕX174 as a model parvovirus and human serum immunoglobulin G (hIgG) as a model protein. The virus retention in 1 g/L hIgG solutions was consistently less than that in a protein-free buffer solution by between 1 to 3 logs for the different virus filters. The virus retention profiles for the two homogeneous membranes were very similar, with the virus retention being highly correlated with the extent of flux decline. Membranes prefouled with hIgG and then challenged with phages also showed much lower virus retention, demonstrating the importance of membrane fouling; the one exception was the Viresolve® Pro membrane, which showed a similar virus retention for the prefouled and pristine membranes. Experiments in which the protein was filtered after the virus challenge demonstrated that hIgG can displace previously captured viruses from within a filter. The magnitude of these effects significantly varied for the different virus filters, likely due to differences in membrane morphology, pore size distribution, and chemistry, providing important insights into the development/application of virus filtration in bioprocessing.