Characterization of apoptosis in PER.C6® batch and perfusion cultures

Mercier, Sarah M.; Diepenbroek, Bas; Martens, Dirk E.; Wijffels, René H.; Streefland, Mathieu

doi:10.1002/bit.25459

Cited by 11 publications

(8 citation statements)

References 56 publications

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“…Similar improvement was shown with a 30% increased recombinant expression yield of the reticulocyte binding protein PfRh5 as antigen for malaria vaccine in S2 cells [64], and 2.3-fold increase in volumetric yields of recombinant influenza vaccines produced in Sf+ cells [115]. Record-breaking appears recent published PER.C6 cell concentrations of 3.6 Â 10 8 cells/ml in alternating tangential flow perfusion systems (ATF, Refine) within 14 days [119]. Newer perfusion systems are based on external hollow-fiber modules with certain molecular weight cut-offs, retaining suspension cells, and optionally viruses, in the cultivation vessel.…”

Section: Process Intensification In Suspension Culturessupporting

confidence: 61%

Bioreactor concepts for cell culture-based viral vaccine production

Gallo-Ramirez

Nikolay²,

Genzel

et al. 2015

Expert Review of Vaccines

124

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Authors contributed equallyVaccine manufacturing processes are designed to meet present and upcoming challenges associated with a growing vaccine market and to include multi-use facilities offering a broad portfolio and faster reaction times in case of pandemics and emerging diseases. The final products, from whole viruses to recombinant viral proteins, are very diverse, making standard process strategies hardly universally applicable. Numerous factors such as cell substrate, virus strain or expression system, medium, cultivation system, cultivation method, and scale need consideration. Reviewing options for efficient and economical production of human vaccines, this paper discusses basic factors relevant for viral antigen production in mammalian cells, avian cells and insect cells. In addition, bioreactor concepts, including static systems, single-use systems, stirred tanks and packed-beds are addressed. On this basis, methods towards process intensification, in particular operational strategies, the use of perfusion systems for high product yields, and steps to establish continuous processes are introduced.

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Section: Process Intensification In Suspension Culturessupporting

confidence: 61%

Bioreactor concepts for cell culture-based viral vaccine production

Gallo-Ramirez

Nikolay²,

Genzel

et al. 2015

Expert Review of Vaccines

124

View full text Add to dashboard Cite

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“…Small nonviable cells are continuously generated in the bioreactor over cultivation time. 50 Moreover, one typically uses up to 2 vessel volumes per day (VVD) perfusion rates for several weeks to months, meaning 2× the volume of the bioreactor is perfused on a daily basis. 24 Therefore, cells in the bioreactor in a perfusion culture will be constantly reprocessed by the viable cell retention/nonviable cell removal system shown in this work, meaning even a marginal removal rate (<30%) can have a significant impact on reducing the number of dead cells present in the bioreactor.…”

Section: Limitation and Potential Use In Perfusion Culturementioning

confidence: 99%

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics

et al. 2018

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Removing nonviable cells from a cell suspension is crucial in biotechnology and biomanufacturing. Labelfree microfluidic cell separation devices based on dielectrophoresis, acoustophoresis, and deterministic lateral displacement are used to remove nonviable cells. However, their volumetric throughputs and test cell concentrations are generally too low to be useful in typical bioreactors in biomanufacturing. In this study, we demonstrate the efficient removal of small (<10 μm) nonviable cells from bioreactors while maintaining viable cells using inertial microfluidic cell sorting devices and characterize their performance. Despite the size overlap between viable and nonviable cell populations, the devices demonstrated 3.5-28.0% dead cell removal efficiency with 88.3-83.6% removal purity as well as 97.8-99.8% live cell retention efficiency at 4 million cells per mL with 80% viability. Cascaded and parallel configurations increased the cell concentration capacity (10 million cells per mL) and volumetric throughput (6-8 mL min −1). The system can be used for the removal of small nonviable cells from a cell suspension during continuous perfusion cell culture operations.

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“…Cell‐specific growth rates ( μ g ) between two sampling points were calculated as ln( X 2 ) − ln( X 1 )/ t 2 − t 1 . X 1 and X 2 representing mean cell densities of two samples, and t 1 and t 2 representing the time points when the two samples were taken . The doubling time was calculated as t = ln2/ μ g .…”

Section: Methodsmentioning

confidence: 99%

Transient gene expression using valproic acid in combination with co‐transfection of SV40 large T antigen and human p21^CIP/p27^KIP

Kiszel¹,

Fiesel²,

Voit³

et al. 2019

Biotechnology Progress

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Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. byco-expression of human cell cycle inhibitors p21 CIP /p27 KIP and Simian virus 40 large T antigen (SVLT). In this study, we investigated the effect of this enhancer protein complex on the TGE experiments in a cell-cycle arrested condition of HEK293F cells induced by valproic acid. Growth profiles, consumptions of nutrients, formations of waste products, and product titers of recombinant human antibodies (huAb) were monitored during the 7-day cultivation time. Our results showed that the use of enhancer proteins increased the product yields in a growth arrest condition as well. During the growth phase, no differences were detected regarding viable cell densities (VCDs), viabilities, growth rates, and cell diameters between the TGE experiments with and without enhancer proteins. However, during the declining phase VCD and viability showed slightly higher values at day 6 and 7 in the presence of enhancers. Furthermore, we could not detect any differences in glucose and glutamine metabolism during batch cultivations with co-expression of enhancer proteins. Taken together, the special complex of enhancer proteins did not contribute to further enhancement of growth arrest and shift in the main cell metabolisms, but resulted in higher cell viability during the decline phase. Our observations suggest that the human cell cycle inhibitors p21 CIP /p27 KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins. K E Y W O R D S antibody production, cell cycle inhibitors, HEK293, SV40 large T antigen, transient gene expression 1 | INTRODUCTION In the process of antibody engineering and high-throughput screening, a simple, rapid, and efficient method of recombinant protein expression is required. Transient gene expression (TGE) in eukaryotic cells is a wellestablished method and has an advantage over stable expression since it has shorter timelines for protein production. 1,2 In the biotechnology industry, the most commonly used mammalian hosts for the production of recombinant proteins are human embryonic kidney (HEK) 293 cells 3 and Chinese hamster ovary (CHO) cells. 4,5 HEK293 cells provide an advantage in the expression of human recombinant proteins due to the pattern of their post-translational modifications. 6 Structure, number and location of post-translational N-glycans are known to affect the biologic activity, stability and half-life of recombinant proteins. 7 To enable cost-effective and efficient high yielding transient transfections, several strategies have been developed. Most of these

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Characterization of apoptosis in PER.C6® batch and perfusion cultures

Cited by 11 publications

References 56 publications

Bioreactor concepts for cell culture-based viral vaccine production

Bioreactor concepts for cell culture-based viral vaccine production

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics

Transient gene expression using valproic acid in combination with co‐transfection of SV40 large T antigen and human p21^CIP/p27^KIP

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Characterization of apoptosis in PER.C6® batch and perfusion cultures

Cited by 11 publications

References 56 publications

Bioreactor concepts for cell culture-based viral vaccine production

Bioreactor concepts for cell culture-based viral vaccine production

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics

Transient gene expression using valproic acid in combination with co‐transfection of SV40 large T antigen and human p21CIP/p27KIP

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Product

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Transient gene expression using valproic acid in combination with co‐transfection of SV40 large T antigen and human p21^CIP/p27^KIP