THE DEVELOPMENT OF THERAPEUTIC MONOCLONAL ANTIBODY PRODUCTS
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Excerpt of Chapter 5

…A critical aspect in the development of any monoclonal antibody product is the preparation of a suitable genetically-modified, engineered cell line capable of producing the product at a sufficiently high titer. Development of the production cell line is among the most important CMC activities because the productivity of the cells will ultimately define the maximum potential overall process yield and will impact all subsequent development activities…

…Most monoclonal antibody products currently in development are expressed in mammalian cell lines with the dominant mammalian cell line used to produce commercial and clinical monoclonal antibody products being the Chinese Hamster Ovary (CHO) cell line. Therefore, the biopharmaceutical industry has focused much effort on developing optimized parental CHO host cell lines, media, and other components that support this specific platform. Many technologies are available to improve the speed of cell line development or the specific productivity levels that can be achieved…

..If the productivity and stability of the initial cell line is high enough, it can be used to support the entire clinical development program as well as commercialization. However, given today’s highly competitive market and the resulting tight development timelines, companies often complete the initial stages of cell line development as quickly and efficiently as possible to enable early entry into human clinical trials with a cell line whose productivity is sufficient at this stage but inadequate to support commercial production. Later in development, sufficient time and resources will then need to be devoted to development of a more optimal cell line that can support late stage and commercial production of the antibody product…

…One of the earliest effective methods for transfection, selection, and amplification of foreign genes in mammalian cells was developed in 1981 by scientists at Columbia University using dihydrofolate reductase (DHFR) selection… The system is effective and has been used in conjunction with the other aspects of cell line development to achieve grams per liter of antibody. However, the total time the DHFR system requires to obtain a suitable production clone (about 1 year total) is often too long to meet the aggressive product development timelines that today’s biopharmaceutical companies strive to achieve….Alternative expression systems that enable development of production cell lines with the desired high antibody expression levels in much shorter times than amplification of DHFR-containing vectors have been developed and are available through individual CMOs or through technology licensing programs. … All of these technologies are considered capable of supporting multi-gram per liter product titers in large-scale cell culture processes and typically have productivities of >30 pg/cell-day in the initial shake flask measurements of individual clones, although the upper limit achievable can be product specific.

… The expression vector of choice is then introduced into the cells using one of many transfection technologies. Following transfection, the cells in which the expression vector DNA was introduced are separated from the non-transfectants by addition of whichever drug or nutrient enables application of selective pressure... To select the production clone from a transfection, various approaches are used to identify 200–500 individual clones for further evaluation. Fluorescence activated cell sorting (FACS) is used by some to identify the higher producing cells, but the selected cells must still be cloned by limiting dilution to obtain single clones for establishment of a production cell line. Other companies select and clone potential cell lines more randomly since the screening process will quickly eliminate low expressers. The 200–500 selected candidate cell lines are grown in small cultures (96 well plates or smaller) and analyzed for antibody productivity using ELISA or other high throughput assays. Using productivity as the initial screening enables scientists to identify 20–100 clones that are then evaluated for growth as well as additional assessments of productivity. At this stage, the selected clones are grown in small shake flasks to determine growth rates, viability, productivity, and gross antibody quality. Based on these assessments, 5–20 clones are expanded into larger shake flask cultures for detailed growth curve and productivity assessment. The final selection of the lead production cell line with up to three back-up lines is normally made by transferring the best candidate cell lines into small bioreactors to determine performance under the conditions of biopharmaceutical production and to begin to measure genetic stability in a fed-batch culture. Clearly the technologies that increase the percentage of transfectants that are high producers and, equally important, are genetically stable throughout many generations would enable companies to screen fewer clones to identify the final production cell line and therefore would reduce timelines and costs.

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