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1. Process- and Product-Related Foulants in Virus Filtration

   https://doi.org/10.3390/bioengineering9040155  

   Isu, Solomon; Qian, Xianghong; Zydney, Andrew L.; Wickramasinghe, S. Ranil (April 2022, Bioengineering)

   Regulatory authorities place stringent guidelines on the removal of contaminants during the manufacture of biopharmaceutical products. Monoclonal antibodies, Fc-fusion proteins, and other mammalian cell-derived biotherapeutics are heterogeneous molecules that are validated based on the production process and not on molecular homogeneity. Validation of clearance of potential contamination by viruses is a major challenge during the downstream purification of these therapeutics. Virus filtration is a single-use, size-based separation process in which the contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filtration is routinely used as part of the overall virus clearance strategy. Compromised performance of virus filters due to membrane fouling, low throughput and reduced viral clearance, is of considerable industrial significance and is frequently a major challenge. This review shows how components generated during cell culture, contaminants, and product variants can affect virus filtration of mammalian cell-derived biologics. Cell culture-derived foulants include host cell proteins, proteases, and endotoxins. We also provide mitigation measures for each potential foulant. 

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2. Single pass tangential flow filtration (SPTFF) for concentration and purification of AAV clarified cell lysate

   https://doi.org/10.1016/j.memsci.2025.124362  

   Chaubal, Akshay S; Yehl, Christopher J; Afzal, Mohammad A; Wang, Lu; Zydney, Andrew L (September 2025, Journal of Membrane Science)

   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. 

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3. Dynamic monitoring and proactive fouling management in a pilot scale gas-sparged anaerobic membrane bioreactor

   https://doi.org/10.1039/d0ew00608d  

   Lim, Kahao; Evans, Patrick J.; Utter, Joshua; Malki, Mo; Parameswaran, Prathap (October 2020, Environmental Science: Water Research & Technology)

   null (Ed.)

   This study examines membrane performance data of a pilot-scale gas-sparged anaerobic membrane bioreactor (AnMBR) over its 472 day operational period and characterizes the foulant cake constituents through a membrane autopsy. The average permeability of 336 ± 81 LMH per bar during the first 40 days of operation decreased by 92% by the study's conclusion. While maintenance cleaning was effective initially, its ability to restore permeability decreased with time. Wasting bioreactor solids appeared to be effective in restoring permeability where chemical cleans were unable to. The restoration mechanism appears to be a decrease in colloidal material, measured by semi-soluble chemical oxygen demand (ssCOD), rather than bioreactor total solids concentration. This is further supported through the use of fluorometry during AnMBR operation, which showed an increase in tyrosine-like compounds during heavy fouling conditions, suggesting that proteinaceous materials have a large influence on fouling. This was corroborated during membrane autopsy using Fourier transform infrared spectroscopy (FTIR). FTIR, scanning electron microscopy with energy dispersive X-ray spectroscopy, and transmission electron microscopy were used to characterize inorganic scalants and predominantly found phosphate salts and calcium sulfate. Fundamentally characterizing foulants and introducing novel and dynamic monitoring parameters during AnMBR operation such as ssCOD and fluorometry can enable more targeted fouling control. 

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4. Host Cell Proteins During Biomanufacturing

   Baik, J.Y.; Guo, J.; Lee, K.H. (October 2019, Current applications of cell culture engineering)

   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. 

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5. Raman imaging of membrane fouling

   https://doi.org/https://doi.org/10.1016/j.seppur.2020.116763  

   Azizighannad, S.; Intrchomb, W.; Mitra, S. (February 2020, Separation and purification technology)

   Membrane processes are widely used in industrial applications such water purification, food processing and pharmaceutical manufacturing. During their operation, the accumulation of foulants in membrane pores and on membrane surfaces lead to the reduction in flux, membrane lifetime and increase in operational cost, and the understanding of the fouling phenomenon is important for mitigating these problems. In this paper we report the application of Raman chemical imaging as a means of identify and map foulants on a membrane surface. The surface of a Polytetrafluoroethylene (PTFE) membrane was studied by Raman chemical imaging before and after fouling during desalination via membrane distillation. Information about location and concentration of three different salts namely CaSO4, BaSO4 and CaCO3 was studied. The three salts showed different distribution patterns, and their distribution was analyzed by correlation mapping and multivariate curve resolution. It was observed that CaSO4 agglomerated in specific places while the BaSO4 and CaCO3 were more distributed. Raman imaging appears to be a powerful tool for studying membrane foulants and can be effective in identifying the distribution of different species on a membrane surface. 

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