Chinese hamster ovary (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins, such as monoclonal antibodies. Enhancement of ...productivity of these CHO cells is one of the top priorities in the biopharmaceutical industry to reduce manufacturing cost. Although there are many different methods (e.g. temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been reported yet. In this study, four different hydrostatic pressures (0, 30, 60, and 90 mmHg) were applied to batch CHO cells, and their cell growth/metabolism and IgG
production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. Moreover, overall IgG
concentration on Day 5 showed that 30 mmHg pressure can increase IgG
production by 26%. The percentage of non-disulphide-linked antibody aggregates had no significant change under pressure. Besides, no significant difference was observed between 30 mmHg and no pressure conditions in terms of cell clumping formation. All these findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.
Continuous production of biologics, a growing trend in the biopharmaceutical industry, requires a reliable and efficient cell retention device that also maintains cell viability. Current filtration ...methods, such as tangential flow filtration using hollow-fiber membranes, suffer from membrane fouling, leading to significant reliability and productivity issues such as low cell viability, product retention, and an increased contamination risk associated with filter replacement. We introduce a novel cell retention device based on inertial sorting for perfusion culture of suspended mammalian cells. The device was characterized in terms of cell retention capacity, biocompatibility, scalability, and long-term reliability. This technology was demonstrated using a high concentration (>20 million cells/mL) perfusion culture of an IgG
-producing Chinese hamster ovary (CHO) cell line for 18-25 days. The device demonstrated reliable and clog-free cell retention, high IgG
recovery (>99%) and cell viability (>97%). Lab-scale perfusion cultures (350 mL) were used to demonstrate the technology, which can be scaled-out with parallel devices to enable larger scale operation. The new cell retention device is thus ideal for rapid perfusion process development in a biomanufacturing workflow.
We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO ...cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Moreover, chemical and mechanical robustness of the nanofluidic device enables continuous monitoring for several days to a week. This continuous and online protein quality monitoring could be deployed at different steps and scales of biomanufacturing to improve product quality and manufacturing efficiency.
Bioprocess optimization towards higher productivity and better quality control relies on real-time process monitoring tools to measure process and culture parameters. Cell concentration and viability ...are among the most important parameters to be monitored during bioreactor operations that are typically determined using optical methods on an extracted sample. In this paper, we have developed an online non-invasive sensor to measure cell concentration and viability based on Doppler ultrasound. An ultrasound transducer is mounted outside the bioreactor vessel and emits a high frequency tone burst (15 MHz) through the vessel wall. Acoustic backscatter from cells in the bioreactor depends on cell concentration and viability. The backscattered signal is collected through the same transducer and analyzed using multivariate data analysis (MVDA) to characterize and predict the cell culture properties. We have developed accurate MVDA models to predict the Chinese hamster ovary (CHO) cell concentration in a broad range from 0.1 × 106 cells/mL to 100 × 106 cells/mL, and cell viability from 3% to 99%. The non-invasive monitoring is ideal for single use bioreactor and the in-situ measurements removes the burden for offline sampling and dilution steps. This method can be similarly applied to other suspension cell culture modalities.
Separation of high‐density suspension particles at high throughput is crucial for many chemical, biomedical, and environmental applications. In this study, elasto‐inertial microfluidics is used to ...manipulate ultra‐high‐density cells to achieve stable equilibrium positions in microchannels, aided by the inherent viscoelasticity of high‐density cell suspension. It is demonstrated that ultra‐high‐density Chinese hamster ovary cell suspension (>26 packed cell volume% (PCV%), >95 million cells mL−1) can be focused at distinct lateral equilibrium positions under high‐flow‐rate conditions (up to 10 mL min−1). The effect of flow rates, channel dimensions, and cell densities on this unique focusing behavior is studied. Cell clarification is further demonstrated using this phenomenon, from 29.7 PCV% (108.1 million cells mL−1) to 8.3 PCV% (33.2 million cells mL−1) with overall 72.1% reduction efficiency and 10 mL min−1 processing rate. This work explores an extreme case of elasto‐inertial particle focusing where ultra‐high‐density culture suspension is efficiently manipulated at high throughput. This result opens up new opportunities for practical applications of high‐particle‐density suspension manipulation.
Elasto‐inertial microfluidics is used to manipulate ultra‐high‐density Chinese hamster ovary cells based on the inherent viscoelasticity of the high‐density suspension. Stable equilibrium positions in microchannels are achieved without the addition of fluid elasticity enhancers. As a practical application, high‐throughput cell clarification is demonstrated, from 29.7 packed cell volume% (PCV%) to 8.3 PCV% with overall 72.1% reduction.
Inertial microfluidics has enabled many impactful high throughput applications. However, devices fabricated in soft elastomer (
, polydimethylsiloxane (PDMS)) suffer reliability issues due to ...significant deformation generated by the high pressure and flow rates in inertial microfluidics. In this paper, we demonstrated deformation-free and mass-producible plastic spiral inertial microfluidic devices for high-throughput cell separation applications. The design of deformable PDMS spiral devices was translated to their plastic version by compensating for the channel deformation in the PDMS devices, analyzed by numerical simulation and confocal imaging methods. The developed plastic spiral devices showed similar performance to their original PDMS devices for blood separation and Chinese hamster ovary (CHO) cell retention. Furthermore, using a multiplexed plastic spiral unit containing 100 spirals, we successfully demonstrated ultra-high-throughput cell clarification (at a processing rate of 1 L min
) with a high cell-clarification efficiency of ∼99% (at the cell density changing from ∼2 to ∼10 × 10
cells mL
). Benefitting from the continuous and clogging-free separation with an industry-level throughput, the cell clarification device could be a critical breakthrough for the production of therapeutic biologics such as antibodies or vaccines, impacting biomanufacturing in general.
Shear stress during bioreactor cultivation has significant impact on cell health, growth, and fate. Mammalian cells, such as T cells and stem cells, in next-generation cell therapies are especially ...more sensitive to shear stress present in their culture environment than bacteria. Therefore, a base knowledge about the shear stress imposed by the bioprocesses is needed to optimize the process parameters and enhance cell growth and yield. However, typical computational flow dynamics modeling or PCR-based assays have several limitations. Implementing and interpreting computational modeling often requires technical specialties and also relies on many simplifications in modeling. PCR-based assays evaluating changes in gene expression involve cumbersome sample preparation with the use of advanced lab equipment and technicians, hampering rapid and straightforward assessment of shear stress. Here, we developed a simple, cell-based shear stress sensor for measuring shear stress levels in different bioreactor types and operating conditions. We engineered a CHO-DG44 cell line to make its stress sensitive promoter EGR-1 control GFP expression. Subsequently, the stressed CHO cells were transferred into a 96 well plate, and their GFP levels (population mean fluorescence) were monitored using a cell analysis instrument (Incucyte®, Sartorius Stedim Biotech) over 24 hours. After conducting sensor characterization, which included chemical induced stress and fluid shear stress, and stability investigation, we tested the shear stress sensor in the Ambr® 250 bioreactor vessels (Sartorius Stedim Biotech) with different impeller and vessel designs. The results showed that the CHO cell-based shear stress sensors expressed higher GFP levels in response to higher shear stress magnitude or exposure time. These sensors are useful tools to assess shear stress imposed by bioreactor conditions and can facilitate the design of various bioreactor vessels with a low shear stress profile.
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•First report on CHO cell-based fluid shear stress sensor in bioprocessing.•Sensor measures overall shear stress using conventional fluorescence microscopy.•Evaluated shear stress levels in commercial bioreactors.•Useful for designing vessels and impellers with low shear profile.
Assessment of airway secretion cells, both for research and clinical purposes, is a highly desired goal in patients with acute and chronic pulmonary diseases. However, lack of proper cell isolation ...and enrichment techniques hinder downstream evaluation and characterization of cells found in airway secretions. Here, we demonstrate a novel enrichment method to capture immune-related cells from clinical airway secretions using closed-loop separation of spiral inertial microfluidics (C-sep). By recirculating the output focusing stream back to the input reservoir and running continuously with a high flow processing rate, one can achieve optimal concentration, recovery and purity of airway immune cells from a large volume of diluent, which was not readily possible in the single-pass operation. Our method reproducibly recovers 94.0% of polymorphonuclear leukocytes (PMNs), with up to 105 PMNs in clear diluted buffer from 50 μL of airway secretions obtained from mechanically ventilated patients. We show that C-sep isolated PMNs show higher neutrophil elastase (NE) release following activation by phorbol 12-myristate 13-acetate (PMA) than cells isolated by conventional mucolytic method. By capturing cells without chemically disrupting their potential function, our method is expected to expand the possibility of clinical in vitro cell based biological assays for various pulmonary diseases such as acute respiratory distress syndrome, pneumonia, cystic fibrosis, and bronchiectasis.
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•Cell cluster removal device based on spiral inertial microfluidics.•Cell cluster removal with high volumetric throughput, removal efficiency, and biocompatibility.•The first ...demonstration of the selective removal of large CHO cell clusters from bioreactors using microfluidics.•The first report on the large-particle trapping effect in spiral inertial microfluidic devices.
Suspension cultures of Chinese hamster ovary (CHO) cells are extensively employed in biopharmaceutical production. During cultivation, CHO cell clusters often form due to cell death and other irregularities, substantially reducing cell growth and productivity. In this study, we introduce a novel method for the selective removal of large CHO cell clusters from bioreactors, utilizing the large-particle trapping effect in spiral inertial microfluidics. A single spiral inertial microfluidic device effectively removed large clusters from CHO cell culture, demonstrating high volumetric throughput, removal efficiency, and biocompatibility. This straightforward, agent-free, and continuous-flow approach can be applied to remove large mammalian cell clusters across various scales and modes of operation (discontinuous or continuous) during cell line development, bioprocess optimization, and biomanufacturing.
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