Perfusion mammalian cell culture for recombinant protein manufacturing – A critical review

Bielser, Jean Marc; Wolf, Marco; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo

doi:10.1016/j.biotechadv.2018.04.011

Cited by 205 publications

(189 citation statements)

References 127 publications

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“…Viable cell densities (VCDs) of up to 130 × 10 6 c/ml and high productivities, as well as space–time yields (STYs) were reported. Despite high promises of this early technology, perfusion processes were rarely implemented in industry because of more complex process control, higher risk of process failure and higher media demands compared to traditional batch or established fed‐batch processes …”

Section: Introductionmentioning

confidence: 99%

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

Mayrhofer

Reinhart

Castan³

et al. 2019

Biotechnology Progress

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Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space–time‐yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone‐specific, high‐performing perfusion media we present a straightforward and rapid two‐step approach combining commercially available basal media and feed supplements using design‐of‐experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small‐scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 106 c/ml within 2 weeks at minimum cell‐specific perfusion rates as low as 10–30 pL/c/d. Obtained STYs of 0.4–1.2 g/L/d represent a 10‐fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.

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Section: Introductionmentioning

confidence: 99%

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

Mayrhofer

Reinhart

Castan³

et al. 2019

Biotechnology Progress

View full text Add to dashboard Cite

show abstract

“…Although the modern biopharmaceutical industry focuses primarily on fed‐batch processes (Shukla & Thömmes, ), perfusion processes are a substantial part of upcoming manufacturing technologies as they can provide multiple advantages. In addition to their economic feasibility (Croughan, Konstantinov, & Cooney, ), continuous processes using cell retention can provide higher cell densities, prolonged cultivation times, both removal of inhibitory by‐products, and continuous substrate addition (Bielser, Wolf, Souquet, Broly, & Morbidelli, ; Voisard, Meuwly, Ruffieux, Baer, & Kadouri, ). Alternating tangential flow (ATF) and tangential flow filtration (TFF) using filtration via hollow fiber modules can potentially reach cell densities of well above 100 × 10 6 cells/ml (Clincke, Mölleryd, Zhang, et al, ) and stable long‐term operation with high viabilities (Warikoo et al, ; Xu, Hoshan, & Chen, ).…”

Section: Introductionmentioning

confidence: 99%

Perfusion cultures require optimum respiratory ATP supply to maximize cell‐specific and volumetric productivities

Becker

Junghans

Teleki

et al. 2019

Biotech & Bioengineering

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Perfusion processes are an emerging alternative to common fed‐batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell‐specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell‐specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.

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“…Cell densities >90E6 were attained for some conditions, with viabilities generally >90%, indicating that the perfusion mimic method used here was a significant improvement over previously reported high throughput methods. 22,23 Current perfusion cell culture process development is trending to higher cell culture densities, 24,25 and these results indicate that the ambr15 perfusion centrifugation model could handle densities in this range.…”

Section: Media Developmentmentioning

confidence: 93%

Development of a novel, high‐throughput screening tool for efficient perfusion‐based cell culture process development

Gagliardi

Chelikani

Yang

et al. 2019

Biotechnology Progress

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Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high‐throughput cell culture tools specifically for perfusion‐based cell culture processes. Here, we have developed a high‐throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high‐throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion‐based cell culture processes.

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Perfusion mammalian cell culture for recombinant protein manufacturing – A critical review

Cited by 205 publications

References 127 publications

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

Rapid development of clone‐specific, high‐performing perfusion media from established feed supplements

Perfusion cultures require optimum respiratory ATP supply to maximize cell‐specific and volumetric productivities

Development of a novel, high‐throughput screening tool for efficient perfusion‐based cell culture process development

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