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1. Enhanced filtration performance using feed‐and‐bleed configuration for purification of antibody precipitates

   https://doi.org/10.1002/btpr.3082  

   Li, Zhao; Chen, Ting‐Hsi; Andini, Erha; Coffman, Jonathan_L; Przybycien, Todd; Zydney, Andrew_L (September 2020, Biotechnology Progress)

   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. 

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2. Development of a local wall concentration model for the design of single pass tangential flow filtration (SPTFF) systems with viral vector surrogates

   https://doi.org/10.1016/j.memsci.2024.123276  

   Chaubal, Akshay S; Single, Alexis J; Zydney, Andrew L (January 2025, Journal of Membrane Science)

   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. 

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3. Addressing amino acid‐derived inhibitory metabolites and enhancing CHO cell culture performance through DOE‐guided media modifications

   https://doi.org/10.1002/bit.28403  

   Ladiwala, Pranay; Dhara, Venkata Gayatri; Jenkins, Jackson; Kuang, Bingyu; Hoang, Duc; Yoon, Seongkyu; Betenbaugh, Michael J. (April 2023, Biotechnology and Bioengineering)

   Abstract Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over‐supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed‐batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed‐batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs. 

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4. Nanoparticle filtration through microporous ECTFE membrane in an alcoholic solution

   https://doi.org/https://doi.org/10.1016/j.seppur.2018.08.022  

   Na Yao, Boris Khusid (October 2018, Separation and purification technology)

   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. 

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5. Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfiltration

   https://doi.org/doi.org/10.1002/btpr.2886  

   Li, Z; Gu, Q; Coffman, JL; Przybycien, T; Zydney, AL (January 2019, Biotechnology progress)

   null (Ed.)

   There is renewed interest in the possibility of using precipitation for initial capture of high-value therapeutic proteins as part of an integrated continuous downstream process. Precipitation is greatly facilitated by the high product titers now achieved in most cell culture processes, in sharp contrast to chromatographic processes whose performance is reduced at high titers. The current study used a combination of reversible cross-linking (zinc chloride, ZnCl2) and volume exclusion (polyethylene glycol) agents to precipitate a monoclonal antibody product directly from harvested cell culture fluid using a continuous tubular precipitation reactor. The precipitates were then dewatered and continuously washed using tangential flow filtration, with a countercurrent-staged configuration used to reduce the amount of wash buffer required and increase host cell protein removal. Long-term operation was achieved by operating the membrane modules below the critical filtrate flux to avoid fouling. Experimental results demonstrate the feasibility of this fully continuous integrated precipitation process at bench scale, with design calculations used to explore the key factors affecting the performance of this system for initial antibody capture. 

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