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

   https://doi.org/doi.org/10/1002/btpr.3082  

   Li, Z; Chen, TH; Andini, E; Coffman, JL; Przybycien, T; Zydney, AL (January 2021, Biotechnology progress)

   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. 

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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. IgG-BSA separation and purification by internally staged ultrafiltration

   https://doi.org/10.1016/j.seppur.2024.129245  

   Feng, Lixin; Song, Yufeng; Basuray, Sagnik; Sirkar, Kamalesh K (August 2024, Separation and Purification Technology)

   Purification of IgG from residual host cell proteins (HCPs) in post-Protein A chromatography is important since some HCPs bind with Protein A and elute with the monoclonal antibody (mAb); removal of HCPs from CHO cell lines is essential. To that end, an advanced separation and purification technique in biopharmaceutical manufacturing, namely, internally staged ultrafiltration (ISUF), is investigated here. Choosing BSA as a model for HCPs in post-protein A eluate, separation of a binary mixture of IgG and BSA containing 1.0 mg/ml IgG and 0.1 mg/ml BSA is successfully demonstrated here using a modified ISUF technique: two Omega 100 kDa membranes on top followed by one Omega 70 kDa membrane at the bottom. This modified configuration demonstrated exceptional performance with almost complete rejection, 99 % purity, and 99.5 % retention of IgG, along with 96.5 % recovery of BSA over 10 diavolumes. This modified membrane stacking resulted from strategic considerations of membrane stacking and careful selection of molecular weight cutoffs and materials, and performance analysis of different membranes and stacking configurations using rejection behaviors, purity levels, and recovery rates under varying diavolume and pressure differential. The approach adopted here enhances flexibility in membrane choices in ISUF and provides valuable insights for optimizing membrane-based biopharmaceutical separation techniques. 

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5. Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

   https://doi.org/10.1002/btpr.3115  

   Zhang, Da; Patel, Parag; Strauss, Daniel; Qian, Xianghong; Wickramasinghe, S_Ranil (January 2021, Biotechnology Progress)

   Abstract Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification. 

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