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).
Figure 2. Antibody Cell Line Development Method.
Pooled Cell Line Production
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.
Figure 3. Process Timeline for Variant Production From a Pooled Cell Line.
Clonal Cell Line Production and Manufacturing
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.
Figure 4. Process Timeline From cDNA to Start of Master Cell Banking Using the GPEx Cell Line Development Method