Flexible Methodology for Developing Mammalian Cell Lines - - BioPharm International

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Flexible Methodology for Developing Mammalian Cell Lines


BioPharm International



Figure 2. Antibody Cell Line Development Method.
GPEx cell lines expressing recombinant proteins and antibodies are produced as shown in Figures 1 and 2. For generation of antibody producing cell lines, an initial transduction of Chinese hamster ovary (CHO) cells is performed using a retrovector containing the light chain (LC) gene. The LC expressing pool of cells is then transduced with a retrovector containing the heavy chain (HC) gene. Upon completing a single transduction, or both LC and HC transductions in the case of antibodies, the resulting pool of cells produces functional protein. These stable pools can be expanded for protein production and analysis. Single cell clones are isolated from the pool using limited dilution cloning. Cells are only cultured in serum-free medium. However, 2% fetal bovine serum is typically used for approximately 10-14 days during the limited dilution cloning step. Cells are routinely cultured at 37 C and 5% CO2. Fed-batch development is completed with a single round of analysis using generic conditions and commercially available media/supplements (HyClone, Logan, UT).

Pooled Cell Line Production


Figure 3. Process Timeline for Variant Production From a Pooled Cell Line.
The ability to produce substantial amounts of protein prior to clonal selection is an advantage of this cell line development method. Stable cell pools producing 20–280 mg/L of over 40 different proteins in standard serum-free overgrowth cultures without feeding have been generated using GPEx. Transfer of the pools to fed-batch conditions results in a 2–4 fold increase over the initial production levels. These production levels allow milligram to multi-gram quantities to be made from 1–100 liter production vessels at this early stage in the cell line development process. A timeline for production of protein variants from pooled cell lines is shown in Figure 3.

Clonal Cell Line Production and Manufacturing


Figure 4. Process Timeline From cDNA to Start of Master Cell Banking Using the GPEx Cell Line Development Method
After completing limited dilution cloning and initial clonal selection on pooled cell lines, top clones are identified and analyzed in generic fed-batch conditions. The top clone is identified from the fed batch analysis and moved into bioreactor manufacturing using the same fed-batch conditions. Results of fed-batch bioreactor runs for nine different antibody producing cell lines are indicated in Table 1. Protein production, specific productivity (picograms/cell/day), and maximum cell density were recorded for each batch. The ability to manufacture clinical material without detailed cell line process development and achieve greater than 1 g/L levels of production dramatically reduces timelines for clinical product production. More detailed process development can then be performed on the cell line while early clinical analysis is being performed. A timeline from gene to master cell bank candidate selection is shown in Figure 4.

Summary


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