Development of mL-scale pseudo-perfusion methodologies for high-throughput early phase development studies

Tregidgo, Molly; Lucas, Ciara; Dorn, Marie; Martina, Micheletti

doi:10.1016/j.bej.2023.108906

Cited by 6 publications

(6 citation statements)

References 36 publications

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“…MWP cultures were performed using standard round well ultra-low attachment 24-well MWP (CLS3473; Corning ® ) and operated using a sacrificial well methodology as previously described by Tregidgo et al (2023). The plates were sealed with a Duetz sandwich lid (CR1524; EnzyScreen) to reduce evaporation while maintaining headspace gas exchange.…”

Section: Mwp Culturesmentioning

confidence: 99%

“…However, several studies evaluating a number of well geometries, shaking speeds, and fill volumes showed that sufficiently high k L a values can be reached to support HCD cultivations (Doig et al, 2005;Hermann et al, 2003;Zhang et al, 2008). For 24-well MWPs, maximum VCC values up to 60 × 10 6 cells mL −1 were reported, as well as good agreement with a 5 L bioreactor in perfusion mode (Tregidgo et al, 2023).…”

mentioning

confidence: 94%

“…Recent studies have investigated 24-well microwell plates (MWP, V W = 1.2 mL) in semi-perfusion using a sacrificial well methodology as a small-scale platform for optimization of perfusion processes (Tregidgo et al, 2023). Similar to spin tubes, oxygen supply becomes critical at HCD for MWP formats.…”

mentioning

confidence: 99%

“…Questions remain regarding the ability to mimic basic perfusion characteristics such as establishing and maintaining a stable environment. It was previously shown that HCD can be achieved in 24-well MWP (Tregidgo et al, 2023), and this study evaluates the platform regarding its capabilities of maintaining HCD at a specific target VCC and thus establishing a quasi-steady state at small scale. In this work this previously established semi-perfusion MWP method was adapted by implementing a cell bleed strategy into the workflow.…”

mentioning

confidence: 99%

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Platform development for high‐throughput optimization of perfusion processes: Part I: Implementation of cell bleeds in microwell plates

Dorn,

Klottrup‐Rees,

Lee

et al. 2024

Biotech & Bioengineering

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The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developing and optimizing perfusion operations requires screening various parameters, which is expensive and time‐consuming when using benchtop bioreactors. Scale‐down models (SDMs) are the most feasible option for high‐throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi‐perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24‐well MWP (Vw = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady‐state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (VW = 30 mL), comparing a squared six‐well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs. SF) and scales (MWP vs. DWP + SF). The results show that the well‐plate‐based methods are suitable to perform HCD and quasi steady‐state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.

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Section: Mwp Culturesmentioning

confidence: 99%

mentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Platform development for high‐throughput optimization of perfusion processes: Part I: Implementation of cell bleeds in microwell plates

Dorn,

Klottrup‐Rees,

Lee

et al. 2024

Biotech & Bioengineering

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“…This is especially advantageous during medium development, cell clone screening, and process development, where the MWP displays a straightforward and simple platform for practical handling and fabrication (Lindstrom & Andersson-Svahn, 2012). Previous studies showed that 24-well MWPs in semi-perfusion, using a total medium exchange once per day, can achieve growth to HCD and are a good representation of a 5 L perfusion bioreactor with a perfusion rate of 1 RV d −1 (Tregidgo et al, 2023). In this study, the MWP platform was evaluated for its ability to maintain different target CSPRs by changing the perfusion rate strategy from a fixed 1 RV d −1 to a CSPR-based regime.…”

Section: Introductionmentioning

confidence: 99%

Platform development for high‐throughput optimization of perfusion processes—Part II: Variation of perfusion rate strategies in microwell plates

Dorn,

Lucas,

Klottrup‐Rees

et al. 2024

Biotech & Bioengineering

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The biopharmaceutical industry is replacing fed‐batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell‐specific perfusion rate (CSPR). To obtain a cost‐productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor‐intensive and time‐consuming experiments, as investigations are performed in lab‐scale perfusion bioreactors. Small‐scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi‐perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi‐perfusion. The CSPR‐based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d−1. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 106 cells mL−1 were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR‐based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.

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Two scale‐down tools for the optimization of perfusion bioreactors for the manufacture of biopharmaceuticals

Lucas,

Blackman,

Rayat

et al. 2024

J Adv Manuf & Process

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As the occurrences of cancer rise and the requirement for biotherapeutics increases, there is a growing demand for cost‐efficient methods to manufacture mammalian cell‐based biopharmaceuticals. Currently, the most cost‐efficient manufacturing solution is the cultivation of cells in perfusion mode. Perfusion allows for high cell densities of up to 200 million viable cells per mL (MVC mL−1) to be achieved, resulting in an increase in yield and volumetric productivity, which is the production of product per unit volume and time. However, culturing in perfusion mode requires large volumes of media, which is a significant expense in process development. Therefore, methods that allow rapid optimization of perfusion media are desirable to decrease operating costs and increase productivity. In this work, a quasi‐perfusion methodology using microwell plates (MWP) is used for media optimization to culture CHO‐S cells producing IgG1 monoclonal antibodies (mAb) known as trastuzumab, which have clinical applications treating HER2+ breast cancer. Results show blending glucose‐rich supplements and sodium butyrate with perfusion‐specific base media can lead to an 8‐fold increase in monoclonal antibody titre compared with traditional fed‐batch media. The original and optimized media were then scaled‐up to a custom made, mini bioreactor (MBR) running in perfusion mode. Two‐fold higher cell density is achieved in the MBR compared with the MWP, however, when normalizing for cell density, mAb productivity is comparable between the two methodologies. The combined MWP and 250 mL MBR methodology is an optimization tool that enables process development cost savings due to reduced volume of media utilization.

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Development of mL-scale pseudo-perfusion methodologies for high-throughput early phase development studies

Cited by 6 publications

References 36 publications

Platform development for high‐throughput optimization of perfusion processes: Part I: Implementation of cell bleeds in microwell plates

Platform development for high‐throughput optimization of perfusion processes: Part I: Implementation of cell bleeds in microwell plates

Platform development for high‐throughput optimization of perfusion processes—Part II: Variation of perfusion rate strategies in microwell plates

Two scale‐down tools for the optimization of perfusion bioreactors for the manufacture of biopharmaceuticals

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