Alleviation of proteolytic degradation of recombinant human bone morphogenetic protein-4 by repeated batch culture of Chinese hamster ovary cells

Kim, Che Lin; Bang, You Lim; Kim, Young Sik; Jang, Ju Woong; Lee, Gyun Min

doi:10.1016/j.procbio.2016.05.007

Cited by 9 publications

(8 citation statements)

References 29 publications

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“…Repeated‐batch cultures were shown to be advantageous for the study of cell cycle dependencies because overlaying effects, such as substrate limitations and presumed metabolite inhibitions could be avoided (Möller et al, ). Furthermore, they offer a semicontinuous cultivation concept for long term studies (Kim, Bang, Kim, Jang, & Lee, ) and are used as a scale‐down model for example perfusion processes (Chen, Wong, & Goudar, ; Wolf et al, ).…”

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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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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“…On Day 2, the rhBMP‐4 concentration in the xylt2‐knockdown cells (0.33–0.36 μg/ml) was approximately 1.2‐fold higher than that in the control cells (Figure b). The decrease in rhBMP‐4 concentration was probably because of cellular uptake and/or proteolytic degradation of rhBMP‐4 in IMDM (Kim et al, ). However, because cell concentration and viability were similar at all three incubations, the lower decrease in rhBMP‐4 observed in xylt2‐knockdown cells was probably due to less internalization of rhBMP‐4.…”

Section: Resultsmentioning

confidence: 99%

“…To further investigate the potential of DS as a chemical additive to improve rhBMP‐4 production, 1.0 g/L of DS was added on Day 0 and 5 to fed‐batch cultures of CHO‐BMP‐4 cells, which was performed as described previously (Kim et al, ). Addition of DS during fed‐batch cultures resulted in an approximately 2.3‐fold increase of the maximum rhBMP‐4 concentration, compared with the control fed‐batch cultures without the addition of DS (Supporting Information Figure S1).…”

Section: Resultsmentioning

confidence: 99%

“…A dihydrofolate reductase‐deficient CHO cell line (DG44) was maintained in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA) supplemented with 7% (v/v) dialyzed fetal bovine serum (dFBS; Sigma‐Aldrich, St. Louis, MO), and 1× hypoxanthine/thymidine (HT) supplement (Gibco, Grand Island, NY). An recombinant CHO (rCHO) cell line producing rhBMP‐4 (CHO‐BMP‐4) was established and was adapted to grow in serum‐free suspension cultures as described previously (Kim et al, ). CD OptiCHO (Invitrogen) supplemented with 8 mM glutamine and 1 µM methotrexate (MTX) was used for the suspension culture.…”

Section: Methodsmentioning

confidence: 99%

“…However, it is difficult to achieve a high titer of rhBMPs in CHO cell culture because there are insufficient quantities of PCs in CHO cells and rhBMPs are prone to degradation in the decline phase of growth. To improve product yield, several strategies, such as PC overexpression and optimization of the cell culture environment, have been attempted with limited success (Kim, Bang, Kim, Jang, & Lee, 2016;Sathyamurthy et al, 2012Sathyamurthy et al, , 2015.…”

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confidence: 99%

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Improving the production of recombinant human bone morphogenetic protein‐4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis

Kim

Jung

Kim

et al. 2018

Biotech & Bioengineering

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Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules, such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP‐4 (rhBMP‐4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP‐4 was incubated with CHO cells, rhBMP‐4 was actively internalized into cells. Cell surface bound heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP‐4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP‐4. In addition, treatment with endocytosis inhibitors (chlorpromazine, genistein, and dynasore) identified a clathrin‐ and dynamin‐dependent endocytic pathway as the major route for rhBMP‐4 internalization. To enhance product yield by minimizing rhBMP‐4 internalization in recombinant CHO (rCHO) cell cultures, we have tested various strategies to reduce HSPG synthesis (knockdown of xylt2 and sodium chlorate treatment) or inhibit the binding of rhBMP‐4 to cell‐surface‐bound HSPGs (supplementation with heparin or dextran sulfate [DS]). Among these approaches, DS, which is a linear anionic sulfated polysaccharide with similarity to HS chains, was the most effective in enhancing rhBMP‐4 production in rCHO cell cultures. Compared with the control cultures, DS addition to the culture medium (1.0 g/L) resulted in 1.4‐fold and 2.3‐fold increases in maximum rhBMP‐4 concentration in batch and fed‐batch cultures, respectively. Taken together, the addition of DS, an effective competitor of HSPGs, improved rhBMP‐4 production in rCHO cell cultures through blockage of rhBMP‐4 internalization.

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BMP-4 Extraction from Extracellular Matrix and Analysis of Heparin-Binding Properties

2021

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Alleviation of proteolytic degradation of recombinant human bone morphogenetic protein-4 by repeated batch culture of Chinese hamster ovary cells

Cited by 9 publications

References 29 publications

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

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

Improving the production of recombinant human bone morphogenetic protein‐4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis

BMP-4 Extraction from Extracellular Matrix and Analysis of Heparin-Binding Properties

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