Cell culture process by intensified perfusion with continuous harvest and without cell bleeding
A cell culture and cell technology, applied in the field of culturing cells and harvesting biological agents, can solve the problems of low yield, difficult to clarify the mixture of cells and biological products, etc.
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Embodiment 1
[0114] In this example, using clone X, the performance of the enhanced perfusion culture process (B) was directly compared to the performance of the conventional fed-batch culture process (A) and the concentrated fed-batch culture process (C).
[0115] Traditional fed-batch culture process A:
[0116] Process A was performed in shake flasks. Traditional fed-batch culture Process A was performed with an initial working volume of 50 mL in a 250 mL vessel volume. In CDM4 medium (Hyclone) supplemented with 4.0 mM L-glutamine, 0.40 × 10 6 cells / mL and cultured for 14 days. In the culture process, 3.00% of the feed medium CB7a and 0.30% of the feed medium CB7b were fed on the 3rd day, the 6th day, the 8th day and the 10th day, respectively. On day 5 the temperature shifted from 36.5°C to 31.0°C. The glucose concentration was maintained at 4.0 g / L by adding a 400 g / kg glucose stock solution throughout the cultivation process.
[0117] Enhanced perfusion culture process B:
[01...
Embodiment 2
[0149] In this example, using clone X, the performance of the intensive perfusion culture process (B) was evaluated.
[0150] 【Enhanced perfusion culture process】
[0151] Experiments IPC-1-IPC-8 were performed using a delta V controller to control temperature at about 36.5°C, pH range between 7.2 and 6.8 and DO at about 40% air saturation. All processes run in ATF flow mode using the ATF-2H system (Refine Technology) used 0.2 μm cut-off hollow fiber filters (Refine Technology) to retain cells except Process 5 which had a cut-off hollow fiber filter with 0.45 μm pore size pores.
[0152] Experiments IPC-1, IPC-2 and IPC-3 were carried out in 7L Applikon containers, and experiments IPC-4, IPC-5, IPC-6, IPC-7 and IPC-8 were carried out in 3L Applikon containers.
[0153] The experimental culture from IPC-1 to IPC-8 starts from about 0.90~1.10×10 in CDM4 medium (Hyclone) supplemented with 4.0 mM L-glutamine 6 cells / mL, and add about 10-100ppm antifoaming agent every day from th...
Embodiment 3
[0163] In this example, using clone Y, the performance of the enhanced perfusion culture process (B) was directly compared to the performance of the traditional conventional fed-batch culture process (A) and the perfusion culture process (C).
[0164] Traditional fed-batch culture process A:
[0165] Procedure A was performed in a shake flask with an initial working volume of 50 mL in a 250 mL vessel volume. In Excell Advanced CHO medium (Sigma) supplemented with 6mM L-glutamine at 0.40×10 6 cells / mL and cultured for 14 days. In the culture process, 3.00% of the basal medium CB7a and 0.30% of the feed medium CB7b were supplemented on the 3rd day, the 6th day, the 8th day and the 10th day, respectively. On day 5 the temperature shifted from 36.5°C to 33.0°C. By supplementing 400g / kg glucose stock solution, the glucose concentration was controlled above 2.0g / L.
[0166] Enhanced perfusion culture process B
[0167] Process B was performed using a delta V controller to contr...
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