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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. 

Abstract Surfactants like polysorbate (Tween®) are commonly used as excipients in the production of monoclonal antibodies and other recombinant proteins. The retention behavior of these excipients in the final ultrafiltration step can be difficult to predict due to the presence of both monomers and micelles. This study examined the retention of polysorbate during ultrafiltration through cellulose and polyethersulfone membranes with nominal molecular weight cutoffs of 10, 30, and 100 kDa. Novel flux stepping experiments were performed to examine the effects of concentration polarization on surfactant transmission. Polysorbate 20 transmission through the 30 kDamembrane was a strong function of the surfactant concentration, decreasing from nearly 100% for a 2.5 mg/L solution to <10% for a 50 mg/L solution due to high retention of the micelles. Polysorbate transmission was lower for the polyethersulfone membrane due to polysorbate adsorption. A simple mathematical model was developed to describe the polysorbate transmission accounting for the effects of concentration polarization as well as the presence of surfactant monomers and micelles. Model calculations were in good agreement with the experimental data, providing a framework for the analysis and design of ultrafiltration/diafiltration processes for biopharmaceutical formulations containing surfactants. 

Abstract Virus filtration is a crucial step in ensuring the high levels of viral clearance required in the production of biotherapeutics produced in mammalian cells or derived from human plasma. Previous studies have reported that virus retention is often reduced in the presence of therapeutic proteins due to membrane fouling; however, the underlying mechanisms controlling this behavior are still not well understood. Experimental studies were performed with a single layer of the commercially available dual‐layer Pegasus^TMSV4 virus removal filter to more easily interpret the experimental results. Bacteriophage ФX174 was used as a model parvovirus, and human immunoglobulin (hIgG) and Bovine Serum Albumin (BSA) were used as model proteins. Data obtained with 5 g/L solutions of hIgG showed more than a 100‐fold reduction in virus retention compared to that in the protein‐free solution. Similar effects were seen with membranes that were pre‐fouled with hIgG and then challenged with ФX174. The experimental data were well‐described using an internal polarization model that accounts for virus capture and accumulation within the virus filter, with the hIgG nearly eliminating the irreversible virus capture while also facilitating the release of previously captured virus. These results provide important insights into the performance and validation of virus removal filters in bioprocessing. 

Abstract Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF‐SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two‐dimensional gel electrophoresis (2D SDS‐PAGE). In addition, a total of 8 HCP were identified using matrix‐assisted laser desorption/ionization‐mass spectroscopy (MALDI‐MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron‐sized particles which could lead to the plugging of the filters. 

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. 

Abstract As upstream product titers increase, the downstream chromatographic capture step has become a significant “downstream bottleneck.” Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever‐increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl₂) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation‐prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl₂to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7–7.5 wt/vol% together with 10 mM ZnCl₂gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.