Mouse Hybridoma Cell Culture in a Protein-Free Medium Using a Bio-Mimicking Fish-Tail Disc Stirred Bioreactor

by Rodolfo Valdés, Hasel Aragón, Marcos González, Daily Hernández, Déborah Geada, David Goitizolo, Williams Ferro, Adelma Pérez, José García, Yordanka Masforrol, Pedro Aguilar, Gabriel Márquez, Maylín LaO, Tatiana González, Yodelis Calvo, Alexander Hernández, Grechen Menéndez, and Andrés Tamayo
Volume 16, Issue 1 (Spring 2017)

Because the Lambda MINIFOR bioreactor provides good mixing of cell culture, nutrients, and gases without any damaging hydrodynamic forces by using a bio-mimicking “fish-tail“ disc stirrer, it can be successfully applied for the cultivation of bacteria and yeast, insect, plant, and mammalian cells. However, reports on its application in mouse hybridoma cell culture using protein-free media is non-existent in the scientific literature. Therefore, this study describes preliminary findings of the Lambda MINIFOR bioreactor suitability in mouse hybridoma cell culture and antibody production using the SP2/O-Ag14-CB.Hep-1 mouse hybridoma cell and the PFHM-II protein-free medium as models. Results verified 2.45 × 106 viable cells/mL as the highest cell concentration, 86% as maximum cell viability, 0.0156/h as the exponential growth rate, 44 h as cell population doubling time, a stable phenotype measured by limiting dilution after 2.5 months, no antibiotic and antifoam requirements, 71.4% of IgG SDS-PAGE purity in the cell culture harvested supernatant, 38.68 ± 22.29 μg/mL, 39.23 ± 10.66 pg/cell/day, up to 99.5% of purity (sample measured by SDS-PAGE and SE-HPLC) after an IgG capture step based on protein A-Sepharose, a low pH incubation, and size-exclusion chromatography, no molecule aggregation, specificity for the CKTCTT epitope (located in the HBsAg “a” determinant), an IgG affinity constant equal to 1.11 × 1010 M-1, and < 78 pg mouse DNA/mg of IgG. In conclusion, this study corroborated a cumulative CB.Hep-1 mAb production of 1.77 g/15 days and validated the usefulness of the Lambda MINIFOR bioreactor in mouse hybridoma cell culture in protein-free media for research applications...

Valdés R, Aragón H, González M, Hernández D, Geada D, Goitizolo D et al. Mouse hybridoma cell culture in a protein-free medium using a bio-mimicking fish-tail disc stirred bioreactor. BioProcess J, 2017; 16(1): 51–64.

Posted online May 8, 2017.