Application of cyrotechnology techniques: a case study for the production, purification and characterization of humanized antibody secreted by NSO transfectoma

Humanized monoclonal antibodies are widely used for diagnosis and treatment of cancer due to their reduced immunogenicity and high specificity. These valuable biopharmaceuticals are produced using mammalian cells which often require serum when cultured in vitro. Since the use of serum involves many ethical, safety and scientific complications, a high-producing NS0 transfectoma which was isolated using Clone Pix FL system, was directly adapted to serum-free growth media (SFGM) in T75 flasks. This transfectoma has been engineered to stably secrete humanized anti-C2 monoclonal antibody (hum-C2 mab) which is highly specific for a colorectal tumor associated antigen. However, the serum-independent NS0 transfectoma had low cell viability when cultured in spinner flasks. Therefore, triple flasks were used instead. The growth characteristics and also hum-C2 mab productivity were comparable between cells cultured in serum-free and serum-supplemented media. Besides that, an automated liquid chromatography system, Äktaprime Plus, was used to purify hum-C2 mab from filtered and concentrated cell culture supernatant since the conventional method of antibody purification is time-consuming, laborious and prone to errors. The high-throughput nature of Äktaprime Plus enabled the purification hum-C2 mab in less than 30 minutes. In addition, the real-time monitoring and the automated fraction collection of Äktaprime Plus eliminated the need for downstream analysis and decreased the risk of spillage or misplacing of fractions containing precious hum-C2 mab. To evaluate the functionality of hum-C2, a conventional antigen-based ELISA could not be used due to the lack of commercially-available purified C2 antigen. As an alternative, a cell-based ELISA was performed using SW1116 cells and even after humanization, the purified hum-C2 mab was still able to bind to the C2 antigen expressed on the surface of SW1116 cells. In summary, the production of NS0 transfectoma in SFGM using triple flasks and the convenient one-step affinity chromatography using Äktaprime Plus resulted in hum-C2 mab being free from exogenous protein contamination especially from bovine polyclonal antibodies found in serum. From the cell-based ELISA, cell binding was observed which confirms the functionality of purified hum-C2 mab after humanization

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 105 clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003% of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of < 1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones, downstream analysis are still vital to determine the ‘actual’ and stable high producer clones