5.2.2. Optimizing factors using response surface methodology
Based on PBD results, four of the most influential factors were selected for further optimization by CCD in DoE software, resulting in thirty experimental runs (Supplementary table 2). The specified composition of model terms (chosen factors) was employed to prepare culture media for each run according to the designed points via BioLector. Besides, constant values of other media components not in the model were supplemented in the media according to the insulin results (mg/ml) in PBD design and SDS-PAGE results. The concentration corresponding to the highest response for less significant variables and -1 level of insignificant factors were supplied. Following the execution of experiments via BioLector, responses were scrutinized via different models. The best model was picked based on model validation parameters documented in the ANOVA table, fit statistic tables, and diagnostic analysis. DoE software generated the perturbation chart, and the effect of each significant independent and dependent variable on response was registered graphically via contour and 3D plots. Finally, DoE software generated four scenarios about optimal points based on the acquired regression equation. Predicted design scenarios with the highest desirability were dissected and compared to each other using Biolector XT microbioreactor in triplicates again. The suggested optimal media was defined as Scenario-3 (13 mg/ml) with a 3.3 deviation and is ready for scale-up optimization.
Acknowledgments
The authors dedicate this manuscript to the memory of Albert E. Dahlberg and Nizar Turker. The authors gratefully acknowledge the use of the services and facilities of the University of Health Science-Validebag DETAUM (Experimental Medicine Research and Application Center).