A DG44 derived CHO cell producing Her2-binding antibody was studied in this article. The basal medium was CD14 (CD14, OPM Biosciences), CD15(CD15, OPM Biosciences) and Dynamis (A26615, Thermofisher) supplemented with 6 mM Glutamine (G8540, Sigma). Feed medium were EfficientFeed B (FEED B, A12456, Thermofisher) and PFF06 (PFF06, OPM Biosciences). The additive was galactose (G5388, Sigma). The compositions of all the used medium were developed by their vendors.
Dynamis
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dynamis is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform specific functions within a controlled environment. The core function of the Dynamis is to facilitate precise and repeatable measurements or operations, as required by the user's research or analytical needs.
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Lab products found in correlation
Glutamine, by Merck Group (1 mentions)
Efficientfeed b, by Thermo Fisher Scientific (1 mentions)
Galactose, by Merck Group (1 mentions)
Anti clumping agent, by Thermo Fisher Scientific (1 mentions)
Peroxidase affinipure f ab 2 fragment goat anti human igg h l hrp, by Jackson ImmunoResearch (1 mentions)
Cd forticho, by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Avantor (1 mentions)
Tmb 3 3 5 5 tetramethylbenzidine solution, by Thermo Fisher Scientific (1 mentions)
Protein a, by GenScript (1 mentions)
Shake flasks, by Corning (1 mentions)
Shake flasks, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Cell boost 7a, by GE Healthcare (1 mentions)
Cellboost 7b, by GE Healthcare (1 mentions)
L methionine sulfoximine msx, by Merck Group (1 mentions)
8 protocols using dynamis
1
CHO Cell Line Produces Anti-HER2 Antibody
2
Optimized Antibody Production in CHO Cells
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An antibody-producing suspension CHO-K1 cell line was used as the model cell line. All basal media and feeds used in this study were proprietary, chemically defined, and serum-free. Before the fed-batch culture, cell lines were passaged every 4 days at a density of 3 × 105 cells/mL in a humidified incubator maintained at 36.5 °C and 5% CO2 with shaking at 120 rpm.
Experiments were performed in 3 L bioreactors (Applikon, Delft, Netherlands) with an initial culture volume of 1.5 L, and 250 mL shake flasks with an initial culture volume of 50 mL. In this study, two processes were performed, which only differed in incubation temperature. The temperature of process 1 was shifted to 33 °C on day 5, while that of process 2 was maintained at 36.5 °C.
The basal media and feeding media used in this study were chemically defined serum-free media. The basal medium was Dynamis, which was purchased from Thermofisher. The feeding media were FM01A and FM01B, which were developed by our laboratory but produced by Merck Sigma (Nantong, China). From the third day, FM01A added 3% of the initial culture weight every day and each FM01B supplement was 1/10 of FM01A. Both FM01A and FM01B did not contain glucose. Glucose was controlled at 6 g/L by feeding 400 g/L of glucose solution daily.
Experiments were performed in 3 L bioreactors (Applikon, Delft, Netherlands) with an initial culture volume of 1.5 L, and 250 mL shake flasks with an initial culture volume of 50 mL. In this study, two processes were performed, which only differed in incubation temperature. The temperature of process 1 was shifted to 33 °C on day 5, while that of process 2 was maintained at 36.5 °C.
The basal media and feeding media used in this study were chemically defined serum-free media. The basal medium was Dynamis, which was purchased from Thermofisher. The feeding media were FM01A and FM01B, which were developed by our laboratory but produced by Merck Sigma (Nantong, China). From the third day, FM01A added 3% of the initial culture weight every day and each FM01B supplement was 1/10 of FM01A. Both FM01A and FM01B did not contain glucose. Glucose was controlled at 6 g/L by feeding 400 g/L of glucose solution daily.
3
ELISA Assay for Antibody Quantification
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Dynamis (catalog no. A2661501), CD FortiCHO (catalog no. A1148301), and anti-clumping agent (catalog no. 0010057AE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). For ELISA method, Invitrogen™ Nunc MaxiSorp™ flat-bottom 96-well plates (catalog no. 44-2404-21) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Protein A (catalog no. GSZ02201) was purchased from GenScript (Piscataway, NJ, USA). Peroxidase AffiniPure F(ab′)2 fragment goat anti-human IgG (H + L) HRP (catalog no. 109-036-088) was purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). ABTS (Diammonium 2,2′-azinobis[3-ethyl-2,3-dihydrobenzothiazole-6-sulfonate]) was ordered from VWR (Tuas, Singapore). TMB (3,3′,5,5′-tetramethylbenzidine) solution (catalog no. 34028) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
4
Stable CHO and HEK293 Cell Line Development
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Via transfection of the desired antibody genes into the same parental CHO k1 GS cell line, we obtained the stable CHO cell lines A and B expressing different recombinant human mAbs (mAb1, mAb2); the two cell lines were used in this study. The HEK293 cells did not contain the target expressed gene.
Proprietary chemically defined basal and feed media were used (Table 1). Dynamis was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Cell Boost 7a/7b was purchased from GE Healthcare (Chicago, IL, USA). SA (purity ≥99%, it is derived from E. coli fermentation method) was purchased from Wuhan Zhongke Optics Valley Green Biotechnology Co., Ltd. (Wuhan, China).
For HEK293 cell culture, we added 4 mM glutamine (Gln) to the basal medium and added 4 mM Gln every two days after. Vial thaw and cell culture expansion were performed for two CHO-K1 and one HEK293 cell lines using shake flasks (Corning Life Sciences, New York, NY, USA) using the Dynamis medium and cells were cultured in a humidified incubator (INFORS AG, Bottmingen, Switzerland) using the standard conditions of 37.0 • C, 6% CO 2 , and incubated at 100-140 rpm. Cells were passaged every 3-4 days prior to N stage fed-batch production.
Proprietary chemically defined basal and feed media were used (Table 1). Dynamis was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and Cell Boost 7a/7b was purchased from GE Healthcare (Chicago, IL, USA). SA (purity ≥99%, it is derived from E. coli fermentation method) was purchased from Wuhan Zhongke Optics Valley Green Biotechnology Co., Ltd. (Wuhan, China).
For HEK293 cell culture, we added 4 mM glutamine (Gln) to the basal medium and added 4 mM Gln every two days after. Vial thaw and cell culture expansion were performed for two CHO-K1 and one HEK293 cell lines using shake flasks (Corning Life Sciences, New York, NY, USA) using the Dynamis medium and cells were cultured in a humidified incubator (INFORS AG, Bottmingen, Switzerland) using the standard conditions of 37.0 • C, 6% CO 2 , and incubated at 100-140 rpm. Cells were passaged every 3-4 days prior to N stage fed-batch production.
5
Fed-batch Production of HEK293 and CHO-K1 Cells
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The fed-batch production experiments for HEK293 and CHO-K1 cells were performed in 250 mL shaker flasks (Corning Life Sciences, New York, NY, USA) with an initial working volume of 50 mL in a humidified INFORS incubator at 37 • C and 6% CO 2 . The agitation was set at 120 rpm. HEK293 cells were seeded at 2 × 10 5 viable cells/mL and CHO-k1 cells were seeded at 5 × 10 5 viable cells/mL in chemically defined basal media. For HEK293 cells, feed media was added at 3% initial culture volume from day 5, and then fed once every two days; SA was added on day 9, 10, 11, 12, and an amount equivalent to 1/4 of the total was added each time. For CHO-K1 Cells, feed media was added at 5% initial culture volume from day 5, and then fed once every two days; SA was added on day 7, 8, 9, 10, and an amount equivalent to 1/4 of the total was added each time. A glucose stock solution was added as necessary.
The basal media was Dynamis (Thermo Fisher Scientific, Waltham, MA, USA), the feed media were cell boost 7a and cell boost 7b (GE Healthy, Chicago, IL, USA)-10:1, respectively.
The basal media was Dynamis (Thermo Fisher Scientific, Waltham, MA, USA), the feed media were cell boost 7a and cell boost 7b (GE Healthy, Chicago, IL, USA)-10:1, respectively.
6
Cultivation and Analysis of Embryonic Human Kidney Cells
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Embryonic human kidney suspension FreeStyle™ 293-F cells (Thermo Fisher Scientific, United States) were grown in FreeStyle™ 293 expression medium (Gibco, United States) or Dynamis™ supplemented with 8 mM L-glutamine (for STR, Gibco, United States) or GlutaMAX™ (for shake flasks, Gibco, United States). Cells were cultured at 37 °C, 8% CO2, and at 137 rpm in shake flasks (Thermo Fisher Scientific, United States) using a Minitron shaker incubator (INFORS HT, Switzerland) with an orbit of 5 cm. The adherent NIH/3T3 murine fibroblast target cells (ATCC CRL-1658) were maintained in Dulbecco’s modified Eagle medium high glucose, pyruvate (DMEM; Gibco, Germany), supplemented with 10% fetal bovine serum (FBS; Gibco, United States) at 37°C in a humidified atmosphere at 5% CO2. Cell number and viability was accessed using a cell counter (anvajo GmbH, Germany).
7
Stable CHO Cell Line Production
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The two glutamine synthetase (GS) CHO cell lines used in this study to produce EGFRvIII antibody C12 were generated from CHO-K1 cells, as described previously [18 (link)], and were adapted to suspension culture, with have the ability to grow in serum-free medium. Briefly, by co-transfecting the desired EGFRvIII antibody C12 gene with GS gene into CHO-K1 cells, adding MSX screening, and performing subcloning, stable CHO cell lines A and B were obtained, which produced human mAb C12 A and mAb C12 B, respectively. Both C12 A and C12 B have a conserved N-glycosylation site at the N297 position.
The basal media and feeding media used in this study were chemically defined serum-free media. The basal medium was Dynamis, which was purchased from Gibco. The feeding media were Cellboost 7a (Cb7a) and Cellboost 7b (Cb7b), which were purchased from GE Healthcare. Each Cb7b supplement was 1/10 of Cb7a. Glucose concentration was tested off-line once a day from day 3, and then 400 g/L glucose stock solution was feed to adjust the glucose concentration to 8 g/L during the cell culture process to supply sufficient amounts of an energy source and cellular components.
The basal media and feeding media used in this study were chemically defined serum-free media. The basal medium was Dynamis, which was purchased from Gibco. The feeding media were Cellboost 7a (Cb7a) and Cellboost 7b (Cb7b), which were purchased from GE Healthcare. Each Cb7b supplement was 1/10 of Cb7a. Glucose concentration was tested off-line once a day from day 3, and then 400 g/L glucose stock solution was feed to adjust the glucose concentration to 8 g/L during the cell culture process to supply sufficient amounts of an energy source and cellular components.
8
Recombinant Protein Expression in CHO Cells
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The recombinant plasmid was transfected into Chinese hamster ovary (CHO)-K1 cells with a neon electrophoresis apparatus under the conditions of 1400 V, 20 ms and 2 pulses. Subsequent to transfection, the cells were incubated in 5 mL 4 mmol/L Gln-containing Dynamis (Gibco) medium that was preheated to 37 °C for two days. They were then inoculated in 96-well plates at 5000 cells/well for three weeks. The cells were screened with 50 μmol/L l -methionine sulfoximine (MSX, Sigma–Aldrich, St. Louis, MO, USA) at 37 °C and cultured in a 7% CO2 incubator for 3 weeks. The highly expressed clones that were grown in 96-well plates were subcultured from 96-well plates to 24-well stationary plates, and then were cultured again in 24-well plates. The volume of each hole was 2 mL, and the culture medium was Dynamis + 25 μmol/L MSX. The culture conditions were 37 °C, 5% CO2, and 220 rpm (thermostatic oscillator). Cells in the 24 deep-hole plates were diluted for 2–4 passages at a density of 0.3–0.5 × 106/mL until the clones adapted to the suspension culture. The clones with the highest expression levels were selected for production and preparation of protein samples.
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