There was no DNA amplification in the negative controls in which the chromosomal DNA of the wild type CHO cell line was used as template. Due to the clone CHO-HAH5 78 exhibited the highest levels of the HAH5 protein measured by ELISA, it was selected for being adapted to suspension culture. The gradual medium change from DMEM plus FCS to SFM4CHO made the cells to detach of the polystyrene surface and successfully adapted

to suspended culture with stirring (Fig. 3A and B). The initial inoculum for scaling up the suspension culture to the volume of 1 l in spinners was 2,5 × 104 cells/mL (Fig. 3C). Two days later, cells increased twofold their concentration and Caspase inhibitor began to grow until reaching more than 3 × 105 cells/mL at day 7. The next day of culture cells decreased their concentration to around 2,5 × 105 cells/mL and became stable until day 10. By day 11, the cell concentration abruptly dropped to almost 1 × 105 cells/mL. Cell viability ranged between 100% and 80% from days 1 to 8. At day 9, cell viability

began to decrease and by the last day of the experiment there was a 40% of cell viability. The results obtained above led us to maintain the suspension culture until day 10, where cell concentration and viability met acceptable values, hence the production of the HAH5 protein could be favored. In this sense, the concentration of the HAH5 protein was measured by ELISA (Fig. 4). The average production of the HAH5 protein by different batches of the clone GSK J4 manufacturer CHO-HAH5 78 in suspension culture was approximately 5,1 μg/mL. There were no significant differences among the individual batches analyzed. The purification process of the HAH5 protein obtained in the culture supernatant was carried out by immunoaffinity chromatography (IC) using a monoclonal Oxalosuccinic acid antibody against the HAH5 protein (Fig. 5). The graphic of absorbance versus time showed a well-defined peak when the elution buffer was applied to the matrix ( Fig. 5A) which could correspond

to the elution of the HAH5 protein. SDS-PAGE and western blot assays revealed that the peak observed after elution in the graphic of absorbance versus time was indeed the elution of the HAH5 protein ( Fig. 5B and C). The immunoreactive band pattern was the same compared to the observed during the transient transfection of HEK-293 cells. The bands corresponding to the precursor protein HAH50 and the subunits HAH51 and HAH52 were detected. A portion of the HAH5 protein was lost in the material not retained to the matrix, which was not observed during the wash of the matrix. The HAH5 protein purified by IC was obtained with more than 95% of purity as estimated by a SDS-PAGE densitometric analysis. The production of the HAH5 protein in a suspension culture system allowed to obtain enough protein to perform immunodetection assays type ELISA with the aim of detecting antibodies against this protein.