title: Quantification of cell lysis during CHO bioprocesses: Impact on cell count, growth kinetics and productivity.
abstract: High cell densities and high viability are critical quality attributes for mammalian bioprocesses. Determination of living and dead cell numbers is nowadays routinely performed by automated image-based cell analyzers or flow cytometry. However, complete lysis of cells is usually neglected by these devices. We present a novel method for robust quantification of lysed cell populations over the course of a CHO bioprocess. The release of lactate dehydrogenase (LDH) and double stranded genomic DNA in culture supernatants were used as markers for cell lysis. We considered the degradation of both markers over cultivation time, which significantly increased the amount of released LDH and DNA. For correct and robust estimation of lysed cell fractions, degradation of both markers over cultivation time was considered, where redundancy of markers allowed data reconciliation. Calculating the number of cells which were subject to complete cell lysis, we could show that this fraction makes up as much as 30% of the total produced biomass and is not described by measurements of image-based analyzers. Finally, we demonstrate that disregarding cell lysis heavily affects the calculation of biomass yields and growth rates and that increasing levels of cell lysis are related to decreased productivity.
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria; CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Vienna University of Technology, Austria.
authors.forename: Tobias
authors.initials: T
authors.lastname: Klein
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria; CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Vienna University of Technology, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Heinzel
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria; CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Vienna University of Technology, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Kroll
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria; CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Vienna University of Technology, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Brunner
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria; CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Vienna University of Technology, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Division Biochemical Engineering, Austria. Electronic address: lukas.neutsch@tuwien.ac.at.
authors.forename: Lukas
authors.initials: L
authors.lastname: Neutsch
mesh_terms: D000818:Animals; D018533:Biomass; D019149:Bioreactors; D016466:CHO Cells / Q000502:physiology*; D002452:Cell Count / Q000379:methods; D049109:Cell Proliferation; D002470:Cell Survival; D003412:Cricetulus; D003470:Culture Media / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*
chemical_list: D003470:Culture Media; D004247:DNA; D007770:L-Lactate Dehydrogenase
keywords: Bioprocess; Cells lysis; Chinese hamster ovary cells; Double strand DNA; Lactate dehydrogenase; Monoclonal antibody; Physiology