An official website of the United States government
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
This content will become publicly available on September 1, 2026
Single pass tangential flow filtration (SPTFF) for concentration and purification of AAV clarified cell lysate
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
- PAR ID:
- 10653218
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Membrane Science
- Volume:
- 733
- Issue:
- C
- ISSN:
- 0376-7388
- Page Range / eLocation ID:
- 124362
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
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
-
null (Ed.)Precipitation can be used for the initial purification of monoclonal antibodies (mAbs), with the soluble host cell proteins removed in the permeate by tangential flow microfiltration. The objective of this study was to examine the use of a feed-and-bleed configuration to increase the effective conversion (ratio of permeate to feed flow rates) in the hollow fiber module to enable more effective washing of the precipitate. Experiments were performed using human serum Immunoglobulin G (IgG) precipitates formed with 10 mM zinc chloride and 7 wt% polyethylene glycol. The critical flux was evaluated as a function of the shear rate and IgG concentration, with the resulting correlation used to predict conditions that can achieve 90% conversion in a single pass with minimal fouling. Experimental data for both the start-up and steady-state performance are in good agreement with model calculations. These results were used to analyze the performance of an enhanced continuous precipitation–microfiltration process using the feed-and-bleed configuration for the initial capture / purification of a mAb product.more » « less
-
Abstract Precipitation can be used for the initial purification of monoclonal antibodies (mAbs), with the soluble host cell proteins removed in the permeate by tangential flow microfiltration. The objective of this study was to examine the use of a feed‐and‐bleed configuration to increase the effective conversion (ratio of permeate to feed flow rates) in the hollow fiber module to enable more effective washing of the precipitate. Experiments were performed using human serum Immunoglobulin G (IgG) precipitates formed with 10 mM zinc chloride and 7 wt% polyethylene glycol. The critical flux was evaluated as a function of the shear rate and IgG concentration, with the resulting correlation used to predict conditions that can achieve 90% conversion in a single pass with minimal fouling. Experimental data for both the start‐up and steady‐state performance are in good agreement with model calculations. These results were used to analyze the performance of an enhanced continuous precipitation–microfiltration process using the feed‐and‐bleed configuration for the initial capture / purification of a mAb product.more » « less
-
Lee, G.M.; Kildegaard, H. Faustrup; Lee, S.Y. (Ed.)Host cell protein (HCP) impurities, endogenous proteins expressed from host cells, can challenge biopharmaceutical manufacturing. Certain HCPs can persist even after downstream purification, leading to adverse impacts on drug stability and potentially, patient safety. Thus, the quantification and control of HCPs is critical. Although many improvements have been made in HCP quantification and control methods, HCP-associated risks cannot be completely eliminated. A better biophysical understanding of Chinese hamster ovary (CHO) HCPs and advancement of monitoring assays will lead to better controlled biopharmaceutical manufacturing. This chapter will discuss (i) current HCP removal processes for various product types, (ii) the impact of residual HCPs on drug efficacy and safety, (iii) HCP quantification and monitoring methods such as proteomics approaches and enzyme-linked immunosorbent assays (ELISA) using anti-HCP antiserum, (iv) HCP control approaches in both upstream and downstream processes, and (v) future directions for effective HCP risk management strategies.more » « less
