Quantification of cell lysis during CHO bioprocesses: Impact on cell count, growth kinetics and productivity

Klein, Tobias; Heinzel, Nicole; Kröll, Paul; Brunner, Matthias; Herwig, Christoph; Neutsch, Lukas

doi:10.1016/j.jbiotec.2015.04.021

Cited by 24 publications

(36 citation statements)

References 39 publications

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“…A time course of DCC L could be shown for the first time for the already published method in CHO fed‐batch process . The results seem plausible and suggest that the method can be applied for fed‐batch processes.…”

Section: Resultsmentioning

confidence: 67%

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Impact of cell lysis on the description of cell growth and death in cell culture

Kröll

Eilers

Fricke

et al. 2016

Engineering in Life Sciences

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The primary task of process development is a process design that guarantees product quality and maximizes product quantity. One part of the process development is the identification of critical process parameters. Especially in cell culture processes, unwanted cell damage as critical process parameter is still challenging in stirred tank reactors and needs therefore to be considered. Nevertheless, this topic and its effects on process performance are currently not well discussed and not verified in literature until now. The process of cell damage or lysis can be defined as the loss of integrity of the cells. For the investigation of this phenomenon, a model‐based designed fed‐batch cultivation with Chinese hamster ovary cells was performed. Besides measurements of viable and dead cell concentration, lysed cell count was determined by DNA measurements. Based on these analytics, different hypotheses, characterizing the cell death, were compared. From this, four main statements could be derived: (i) consideration of lysis in cell culture processes is of great importance for the description of the living biomass population in terms of growth and dying; (ii) a higher effort in process monitoring facilitates significantly model development; (iii) in contradiction to existing models from literature, our verification approach demonstrated a direct correlation of lysis with viable cell concentration and therefore with productivity; and (iv) lysis could be effectively described by only one model parameter, the specific lysis rate from viable cells to lysed cells. All these statements could be accurately proven by statistical methods. Our results enable future detailed investigations of the causes of cell damage and the influences of cell lysis on product quality and quantity. Potential application fields are scale‐up issues, process optimization, and impurity screening.

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Section: Resultsmentioning

confidence: 67%

“…All measurements were performed in triplicates. The necessary parameters were determined in prior experiments . The intracellular DNA content was determined to 6.2 pg/cell, which is in accordance with published values .…”

Section: Methodsmentioning

confidence: 67%

Impact of cell lysis on the description of cell growth and death in cell culture

Kröll

Eilers

Fricke

et al. 2016

Engineering in Life Sciences

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“…168 –216 hr) and death phase ( t

>

216 hr). The mechanisms of cell death, including apoptosis, necrosis, and cell lysis are not understood properly, even in a holistic way (Klein et al, ; Tabas & Ron, ). Moreover, the influence of these effects on the cell cycle is still unknown and was not targeted in this study (Chaiboonchoe et al, ; Hydbring, Malumbres, & Sicinski, ).…”

Section: Resultsmentioning

confidence: 99%

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Möller

Bhat

Riecken

et al. 2019

Biotech & Bioengineering

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The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody‐producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle‐derived population dynamics. In this study, process‐induced cell cycle synchronization was assessed in repeated‐batch and fed‐batch cultures. An automated flow cytometry set‐up was developed to measure the cell cycle distribution online, using antibody‐producing CHO DP‐12 cells transduced with the cell cycle‐specific fluorescent ubiquitination‐based cell cycle indicator (FUCCI) system. On the basis of the population‐resolved model, feeding‐induced partial self‐synchronization was predicted and the results were evaluated experimentally. In the repeated‐batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed‐batch process with up to 25×106 cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high‐frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle‐dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.

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“…As a comparative method for cell lysis determination, DNA was quantified using the Picogreen assay . The calibration range was established to be 52.5 ng mL −1 to 840 ng mL −1 (Figure 3 A).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, lysed cells cannot be directly detected via cell counting methods. Thus, they are usually indirectly measured through the detection of the released internal constituents, such as DNA, lactate dehydrogenase, or via the detection of cell debris . The change in rheological characteristics represents an additional opportunity to account for cell lysis; however, this is only applicable for microbial systems or high cell density cell culture systems.…”

Section: Introductionmentioning

confidence: 99%

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

Sissolak

Zabik²,

Saric³

et al. 2019

Biotechnology Journal

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Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants. The reliability and reproducibility of the method are tested in a long‐term study and compared with lysis quantification via the DNA measurement. The results show that its performance is more robust and accurate over time and the respective concentration range. Additionally, both methods are used for cell lysis process monitoring in a recombinant Chinese hamster ovary fed‐batch process. In the presented process, by applying the established assay, the lysis rate k DL is determined to be constant over time at 4.6 × 10 −4 lysed cell concentration (LCC) per TCC and time (LCC/TCC/h). In contrast, DNA data did not confirm the constant lysis rate due to variations of the content per cell during cultivation. Thus, information on the RPC can facilitate the determination of the optimal harvest time point with respect to purity and in improving process characterization.

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Quantification of cell lysis during CHO bioprocesses: Impact on cell count, growth kinetics and productivity

Cited by 24 publications

References 39 publications

Impact of cell lysis on the description of cell growth and death in cell culture

Impact of cell lysis on the description of cell growth and death in cell culture

Process‐induced cell cycle oscillations in CHO cultures: Online monitoring and model‐based investigation

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

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