Scale-Down Perfusion Process for Recombinant Protein Expression

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.

Section: Discussionmentioning

confidence: 99%

Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96‐well plates by facilitating oxygen transfer

Meyer

Condon

Keil

et al. 2011

“…The method successfully predicted the increase in t-PA production at lower temperatures though the authors expressed the need to exercise caution when interpreting the results from such a system to avoid erroneous conclusions regarding actual productivity and growth characteristics. 21 To mimic perfusion cultivation at an even smaller scale, shake flasks with a 30 mL working volume have been used to simulate perfusion by centrifuging the suspension every day of the culture and resuspending the cells with fresh media. Although useful as an indicative screening tool, this mimic has failed to reproduce media optimization results obtained in 4 L lab scale bioreactor, most likely because of the lack of pH and DO control in shake flasks.…”

Section: Perfusion Scale-down Modelsmentioning

confidence: 99%

“…Spinner flasks with 100 mL working volume have also been used in a semicontinuous mode to create pseudo‐steady states and study the effect of temperature shifts on tPA (tissue plasminogen activator) producing CHO cells. The method successfully predicted the increase in t‐PA production at lower temperatures though the authors expressed the need to exercise caution when interpreting the results from such a system to avoid erroneous conclusions regarding actual productivity and growth characteristics …”

Section: Introductionmentioning

confidence: 99%

A novel scale‐down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Kreye

Stahn

Nawrath

et al. 2019

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large‐scale perfusion cultures, suitable scale‐down mimics are needed which allow high‐throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed‐batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale‐down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 107 cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large‐scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80‐fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5‐fold compared to a similar experimental setup at bench scale.

“…We performed a two week TS‐simulated HCD where daily medium exchanges mimic the medium replacement rate in 2L BR. Previous reports also described processes with medium exchange in shake flasks, 24 deep‐well plates or TS, but did not comprehensively address the comparability to an established bench‐scale BR, including product quality attributes. Characterization of both growth and product quality in several cell lines is critical to determine when to use a simulated HCD, and how to interpret results to accurately predict performance in instrumented BR.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Tubespins as a suitable scale‐down model of bioreactors for high cell density CHO cell culture

Gómez

Ambhaikar

Zhang

et al. 2017

High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490-499, 2017.