An official website of the United States government
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.
more »
« less
Development of a local wall concentration model for the design of single pass tangential flow filtration (SPTFF) systems with viral vector surrogates
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.
more »
« less
- PAR ID:
- 10558208
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Membrane Science
- Volume:
- 713
- Issue:
- C
- ISSN:
- 0376-7388
- Page Range / eLocation ID:
- 123276
- Subject(s) / Keyword(s):
- Ultrafiltration SPTFF Critical flux Viral vectors AAV Lentivirus
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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.more » « less
-
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.more » « less
-
One major challenge in the development of nanoparticle-based therapeutics, including viral vectors for the delivery of gene therapies, is the development of cost-effective purification technologies. The objective of this study was to examine fouling and retention behaviors during the filtration of model nanoparticles through membranes of different pore sizes and the effect of solution conditions. Data were obtained with 30 nm fluorescently labeled polystyrene latex nanoparticles using both cellulosic and polyethersulfone membranes at a constant filtrate flux, and both pressure and nanoparticle transmission were evaluated as a function of cumulative filtrate volume. The addition of NaCl caused a delay in nanoparticle transmission and an increase in fouling. Nanoparticle transmission was also a function of particle hydrophobicity. These results provide important insights into the factors controlling transmission and fouling during nanoparticle filtration as well as a framework for the development of membrane processes for the purification of nanoparticle-based therapeutics.more » « less
-
Organic solvent filtration is an important industrial process. It is widely used in pharmaceutical manufacturing, chemical processing industry, semiconductor industry, auto assembly etc. Most of the particle filtration studies reported in open literature dealt with aqueous suspension medium. The current work has initiated a study of cross-flow solvent filtration behavior of microporous ethylene chlorotrifluoroethylene (ECTFE) membranes using 12 nm silica nanoparticles suspended in an aqueous solution containing 25% ethanol. In the constant pressure mode of operation of cross-flow microfiltration (MF), permeate samples were collected at different time intervals. The permeate particle size distribution (PSD) results for different experiments were identical. Particle agglomerates having less than 100 nm size can pass through the membrane; some fouling was observed. The governing fouling mechanisms for tests operated using 3.8×10−3 kg/m3 (3.8 ppm) at 6.9×103 Pag and 1.4×104 Pag were pore blocking. For tests conducted using 3.8×10−3 kg/m3 (3.8 ppm) at 27.6×103 Pag (4 psig) and 1.9×10−3 kg/m3 (1.9 ppm) at 6.9×103, 13.8×103 and 27.6×103 Pag (1, 2 and 4 psig), the mechanism was membrane resistance control. Less particles got embedded in membrane pores in experiments operated using suspensions with lower or higher particle concentrations with a higher transmembrane pressure. This is in good agreement with the values of the shear rate in the pore flow and scanning electron microscope images of the membrane after MF. In the dead-end mode of operation of solvent filtration using methanol, ethanol and 2-propanol, the permeate flux behavior follows Jmethanol > Jethanol > J2-propanol at all testing pressures. The values of permeance (kg/m2-s-Pa) determined from the slope of the linear plot of filtration flux vs. the applied pressure difference across the membrane, were 3.9×10−4, 2.3×10−4 and 3.0×10−5 for methanol, ethanol and 2-propanol, respectively. Further exploration was made on solvent sorption results reported earlier. The critical temperature of selected solvents shows a better correlation with solvent sorption rather than the solubility parameter.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1016/j.memsci.2024.123276
