Production of MAbs in Protein Free Medium

1. In this section, we describe our experience of selecting a hybridoma clone capable of secreting high MAb concentrations when growing in a serum-, protein-free medium. See Chapter 13 for a detailed description of the uses of this MAb for the detection of MDR1 P-glycoprotein expression and functional activity.

2. A subclone of the UIC2 hybridoma, termed UIC2/A (ATCC; cat. no. HB11287), was developed by gradually replacing the original growth medium (DMEM supplemented with 10% FBS and penicillin-streptomycin) with PFHM II protein-free hybridoma medium (12). Complete DMEM-based medium was replaced by PFHM II supplemented by 10% FBS during the fourth round of recloning, according to the timeline described in Subheading 3.5.

3. Subsequently, the hybridoma was cultured in 25-mL flasks on complete PFHM II medium. The concentration of FBS was gradually reduced to 0% by replacing 25% of complete PFHM II medium with unmodified PFHM II. After 1 mo of cultivation in the protein-free medium, all cells had lost their ability to grow while attached to the flask surface, and were growing in PFHM II as a suspension culture in clusters consisting of 8-20 cells.

4. At that point, hybridoma cells were cloned by serial dilution in unmodified PFHM II in 96-well plates (see Subheading 3.5.), and supernatants from the best wells were tested for antibody production by indirect immunofluorescence. A clone that gave the strongest immunofluorescence signal was selected, transferred to a 25mL flask, and the cell line was further expanded.

5. This cell line, designated UIC2/A (ATCC; cat. no. HB11287), was then passaged as a suspension culture in PFHM II medium in 25-mL, to 75-mL, and, finally, to 175-mL flasks. When supplemented with 25 mM HEPES, UIC2/A could be then cultivated in roller bottles, which resulted in high antibody titers (0.2-0.4 mg/mL).

6. As repeatedly verified by indirect immunofluorescence, the UIC2/A hybridoma produced MAb with the specificity and isotype (IgG2a) identical to those produced by the parental UIC2.

7. The data shown in Fig. 1 demonstrates that the UIC2 MAb present in UIC2/A supernatants was about 80% pure IgG without any purification (lanes 6-9). After a single-step purification on a protein-A affinity column, the purity of the UIC2/A MAb was close to 100% (lanes 3 and 4), similar to that of the protein A-purified MAb from ascitic fluids produced by parental UIC2 hybridoma (lanes 1 and 2). The concentration of the UIC2 antibody in UIC2/A supernatants varied between 200 and 350 p,g/mL, based on the yield determined after affinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

8. The ability of the UIC2/A cell line to grow in suspension (including permanent rotation in roller bottles), the high titers of the UIC2 MAb produced, and the small amount of extraneous proteins make this cell line particularly useful for industrial-scale production of the UIC2 MAb. Additionally, in many applications (e.g., immunofluorescent and Immunohistochemistry (IHC) staining of cells and tissues, cell separation, immunoprecipitation, and so on) UIC2/A supernatant fluids may be used without purification or concentration.

9. When licensed to Chemicon International (Temecula, CA) and manufactured as a commercial research reagent, gram amounts of the UIC2/A antibody were readily produced using the INTEGRA (Chur, Switzerland) cell line perfusion system and the Cellex (Minneapolis, MN) ACUSYST-miniMAX bioreactor.

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