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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 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. 

Abstract Tangential flow filtration (TFF) has many advantages for bioreactor harvesting, as the permeate could be introduced directly to the subsequent capture step. However, membrane fouling has limited its widespread use. This is particularly problematic given the high cell densities encountered today. Here, a reverse asymmetric membrane, where the more open surface faces the feed stream and the tighter 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 yeast suspensions has been conducted under a range of conditions. The yeast cells are trapped in the open pore structure. The membrane stabilizes an internal porous cake that acts like a depth filter. This stabilized cake layer can remove particulate matter that would foul the barrier layer if it faced the feed stream. As filtration continues, a surface cake layer forms on the membrane surface. A resistance in series model has been developed to describe the permeate flux during TFF. The model contains three fitted parameters which can easily be determined from constant pressure normal flow filtration experiments and total recycle constant flux TFF experiments. The model can be used to estimate the capacity of the filter for a given feed stream. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor harvesting. 

Tangential flow microfiltration is easily adapted for batch and continuous bioreactor clarification. The permeate can be introduced directly to the subsequent capture step. However, the commercial use of tangential flow filtration (TFF) is limited by membrane fouling, leading to a compromised performance. Here, we explored the possibility of reducing membrane fouling by integrating a hydrocyclone as the primary clarification operation. The overflow from the hydrocyclone was introduced directly as the feed to the microfiltration module. Chinese hamster ovary cells were used as the feed stream to investigate the feasibility of this integrated process. A range of cell viabilities from 0% (cell lysate) to 96% were investigated. The cell densities ranged from 0.9 to 10 million cells per mL. Two commercially available hollow fiber microfiltration membranes were used, an essentially symmetric membrane and a reverse asymmetric membrane where the more open support structure faced the feed stream. The reverse asymmetric membrane was more resistant to fouling in the absence of an integrated hydrocyclone. Integrating a hydrocyclone led to a reduction in the flux decline for the symmetric membrane, but did not affect the performance of the reverse asymmetric membrane. The careful choice of membrane morphology and pore size is important when designing an integrated process. 

Virus filtration is used to ensure the high level of virus clearance required in the manufacture of biopharmaceutical products such as monoclonal antibodies. Flux decline during virus filtration can occur due to the formation of reversible aggregates consisting of self-assembled monomeric monoclonal antibody molecules, particularly at high antibody concentrations. While size exclusion chromatography is generally unable to detect these reversible aggregates, dynamic light scattering may be used to determine their presence. Flux decline during virus filtration may be minimized by pretreating the feed using a membrane adsorber in order to disrupt the reversible aggregates that are present. The formation of reversible aggregates is highly dependent on the monoclonal antibody and the feed conditions. For the pH values investigated here, pretreatment of the feed using a hydrophobic interaction membrane adsorber was the most effective in minimizing flux decline during virus filtration. Ion exchange membranes may also be effective if the monoclonal antibody and membrane are oppositely charged. Consequently, the effectiveness of ion exchange membrane adsorbers is much more dependent on solution pH when compared to hydrophobic interaction membrane adsorbers. Size based prefiltration was found to be ineffective at disrupting these reversible aggregates. These results can help guide the development of more effective virus filtration processes for monoclonal antibody production. 

In pursuit of sustainability, we explored replacing conventional dissolved air floatation (DAF) in poultry processing wastewater (PPW) treatment with a precisely tuned 0.02 µm stainless-steel ultrafiltration (SSUF) membrane. SSUF is a robust, homogenously porous membrane with strong chemical resistance, ease of cleaning, and exceptional resistance to organic fouling. Unlike polymeric membranes, it can be regenerated multiple times, making it a cost-effective choice due to its compatibility with harsh chemical cleaning. The PPW used for the study was untreated wastewater from all processing units and post-initial screening. Our study revealed the SSUF membrane’s exceptional efficiency at eliminating contaminants. It achieved an impressive removal rate of up to 99.9% for total suspended solids (TSS), oil, grease, E. coli, and coliform. Additionally, it displayed a notable reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD), and total Kjeldahl nitrogen (TKN), up to 90%, 76%, and 76%, respectively. Our investigation further emphasized the SSUF membrane’s ability in pathogen removal, affirming its capacity to effectively eradicate up to 99.99% of E. coli and coliform. The measured critical flux of the membrane was 48 Lm−2h−1 at 38 kPa pressure and 1.90 m/s cross-flow velocity. In summary, our study highlights the considerable potential of the SSUF membrane. Its robust performance treating PPW offers a promising avenue for reducing its environmental impact and advocating for sustainable wastewater management practices. 

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.