Critical phases of viral production processes monitored by capacitance

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real‐time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell‐baculovirus expression vector system (IC‐BEVS), a well‐established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real‐time monitoring tool for recombinant adeno‐associated virus (rAAV) production in the IC‐BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm‐based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave‐one‐batch‐out cross‐validation. Our approach shows fluorescence spectroscopy allows real‐time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC‐BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno‐associated virus production by fluorescence spectroscopy

Pais

Portela

Carrondo

et al. 2019

“…The use of dielectric properties for monitoring biomass is of interest for biopharmaceutical production as the methods are label-free and noninvasive and have the potential to perform online analysis (Carvell & Dowd, 2006;Downey, Graham, Breit, & Glutting, 2014;Kiviharju et al, 2007;Lee, Carvell, Brorson, & Yoon, 2015;Petiot, Ansorge, Rosa-Calatrava, & Kamen, 2017). It has been demonstrated that dielectric spectroscopy is a useful technique to monitor the biomass as well as the membrane and cytoplasm properties of Chinese hamster ovary (CHO) cells (Ansorge, Esteban, & Schmid, 2007;Ansorge et al, 2010a;Cole et al, 2015;Downey et al, 2014;Elias, Zeiser, Bédard, & Kamen, 2000;Justice et al, 2011;Opel et al, 2010).…”

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confidence: 99%

“…Dielectric spectroscopy techniques use the average dielectric properties of the cell culture medium to estimate the concentration of cells and their dielectric properties (Ansorge et al, 2010a(Ansorge et al, , 2010bAsami, Takahashi, & Takashima, 1989;Asami & Yamaguchi, 1992;Cannizzaro et al, 2003;Cole et al, 2015;Courtès et al, 2016;Druzinec et al, 2012;Grein et al, 2018;Justice et al, 2011;Katsumoto et al, 2008;Opel et al, 2010;Ron et al, 2008). Employing dielectric spectroscopy, also known as capacitance measurement, for real-time in situ monitoring of the biomass content in fed-batch cultures has been demonstrated to be successful in the estimation of dielectric properties and hence the physiological state of the cells (Ansorge et al, 2010a(Ansorge et al, , 2010bPetiot et al, 2017). The assessment of individual cell properties and identification of subpopulations such as viable and nonviable from bulk dielectric spectroscopic data is challenging as the measured information is related to the overall properties of the cell suspension.…”

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confidence: 99%

Cytoplasmic conductivity as a marker for bioprocess monitoring: Study of Chinese hamster ovary cells under nutrient deprivation and reintroduction

Fazelkhah

Afshar

Braasch

et al. 2019

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed‐batch cultures. The activity level of Na+/K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48‐hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+/K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient‐deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis

Gränicher

Babakhani

Göbel

et al. 2021

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 10 11 TCID 50 /L bioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.semi-continuous production, steric exclusion chromatography, suspension cell culture in bioreactor, upstream and downstream processing, viral vector | INTRODUCTIONTo date, the implementation of an integrated perfusion process is an option to decrease manufacturing costs and to potentially increase the quality of a product (Bielser et al., 2018;Walther et al., 2015Walther et al., , 2019Xu & Chen, 2016). A considerable amount of research has been conducted on the integrated continuous production of recombinant pro-