Investigating and modelling the effects of cell lysis on the rheological properties of fermentation broths

Newton, Joseph; Vlahopoulou, J; Zhou, Yuhong

doi:10.1016/j.bej.2017.01.009

Cited by 14 publications

(18 citation statements)

References 39 publications

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“…By 14 h (which corresponded to the onset of O 2 limitation in the bioreactor), a significant increase in viscosity was observed and the samples also became increasingly shear-thinning. This type of behavior has been described in previous studies of a variety of fermentation processes [34,35,53]. Similar rates were used in this study, but a unique attribute of this work is the viscosity of samples began to increase at a certain shear rate, which was consistent across the different measurements for the individual samples and increased with increasing sample viscosity (and hence cultivation time).…”

Section: Rheological Characterization Of the Cultivation Mediumsupporting

confidence: 84%

“…The maximum concentrations (at 27 h) of proteins, PHA, and DNA in the medium were 1.56 g•L −1 , 0.67 g•L −1 , and 0.49 g•L −1 , respectively. Collectively, the components could account for at most 48% of the total VS detected in the supernatant, which reached a maximum of 9 g•L −1 by 27 h. Newton et al [35] demonstrated that both protein (0-50 g•L −1 ) and DNA (0-4 g•L −1 ) contributed linearly to increased viscosity and the flow curves for solutions of protein and DNA exhibited shear thinning and Newtonian behavior, respectively. However, in that work a somewhat lesser increase in viscosity was noted (1.1 to 5.3 mPa•s) for a 48 g•L −1 E. coli culture, despite the presence of far more extracellular DNA (typically 3 g•L −1 ) and protein (up to 40 g•L −1 ).…”

Section: Quantifying Components Of the Extracellular Matrixmentioning

confidence: 99%

“…However, many of these assessments examined rheological properties of an extracted polymer of interest, but did not directly quantify its effect on culture medium. Most studies show that fermentation medium behaves as a non-Newtonian fluid, meaning the apparent viscosity is dependent on the shear rate [19,23,34,35].…”

Section: Introductionmentioning

confidence: 99%

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Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

et al. 2019

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The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L−1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s−1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L−1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to this behavior, the oxygen transfer performance of the bioreactor, for otherwise constant operating conditions, was reduced by 50% over the cultivation time. This study has shown that the dynamic rheology of HCD cultures is an important engineering parameter that may impact the final outcome in PHA cultivations. Understanding and anticipating this behavior and its biochemical origins could be important for improving overall productivity, yield, process scalability, and the efficacy of downstream processing unit operations.

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Section: Rheological Characterization Of the Cultivation Mediumsupporting

confidence: 84%

Section: Quantifying Components Of the Extracellular Matrixmentioning

confidence: 99%

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Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

et al. 2019

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“…18 Then, the adalimumab Fab analog was expressed and assembled in the periplasmic space of engineered strains under high-density fermentation, according to the reported methods. 19,20 After fermentation, the culture medium of the engineered E. coli strains was centrifuged at 10,000 Â g for 10 minutes at room temperature (r.t.). The microbial pellet was resuspended in five volumes of the suspension buffer (60 mmol/L citric acid and 50 mmol/L MgSO 4 , pH ¼ 4.0).…”

Section: Construction Expression and Purification Of The Adalimumab Fab Analogmentioning

confidence: 99%

Extending the in vivo Half-Life of Adalimumab Fab via Sortase A-Mediated Conjugation of Adalimumab Fab with Modified Fatty Acids

Zhang

Ruan

et al. 2020

Pharmaceutical Fronts

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Adalimumab, a full-length monoclonal antibody, is widely used as an anti-tumor necrosis factor-α (anti-TNF-α) agent. In this article, we aimed to prolong the in vivo half-life of adalimumab antigen-binding fragment (Fab) through Sortase A (SrtA)-mediated conjugation of its Fab with fatty acid (FA). In our study, adalimumab Fab analog was prepared by adding an SrtA recognition sequence (LPETGG) and His6 tag to the heavy chain C-terminal of the Fab via (G4S)3 linker. Four FA motifs with different linkers were designed and synthesized by solid-phase methodology, then conjugated with the Fab analog using SrtA to produce Fab bioconjugates. The successful generation of four Fab bioconjugates (Fab–FA1, Fab–FA2, Fab–FA3, and Fab–FA4) was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and mass spectrometry. Then, the bioactivities and half-life of these Fab bioconjugates were examined using TNF-α-/human serum albumin (HSA)-binding enzyme-linked immunosorbent assay, cytotoxicity assay, and pharmacokinetic study, respectively. All Fab bioconjugates exhibited similar TNF-α-neutralizing activities when compared with the Fab analog, even in the presence of albumin, indicating that there were no apparent influences on the functional site of Fab after FA modification. However, different degrees of affinity for HSA were observed among these Fab–FA bioconjugates, with Fab–FA3 exhibiting the maximal affinity. An in vivo study in mice further revealed remarkably improved pharmacokinetics of Fab– FA3 with a 15.2-fold longer plasma half-life (19.86 hours) compared with that of the Fab analog (1.31 hours). In summary, we have developed a novel long-acting adalimumab Fab bioconjugate, Fab–FA3, with more sustained in vivo activity, which can be used for drug development targeting TNF-α-mediated inflammatory diseases.

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“…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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Investigating and modelling the effects of cell lysis on the rheological properties of fermentation broths

Cited by 14 publications

References 39 publications

Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

Extending the in vivo Half-Life of Adalimumab Fab via Sortase A-Mediated Conjugation of Adalimumab Fab with Modified Fatty Acids

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

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