Development and Validation of a Phenomenological Model for Scalable Size-Exclusion Chromatography in TNF-Blocking Peptide Purification

Resumen

A recombinant peptide as a TNF-blocking agent was obtained using fermentation in E. coli. The strategy was based on the expression of a multimeric tandem protein comprising 15 repeated copies of the r-CBB288 peptide, and the monomeric unit was released with a hydroxylamine-assisted proteolysis step. At this point, size exclusion chromatography was used to separate the monomer unit from unreacted polymer forms. Therefore, the main theme of this study was to develop a phenomenological General Rate Model to describe the elution profile of SEC and to ensure the required purity levels for the mentioned peptide. In that sense, with the development of the phenomenological model, it will be possible to design different process scales of desired dimensions further. For such purpose, the reproducibility of the SEC was determined on a lab scale, and phenomenological coefficients were calculated from mathematical correlations and simulated in the Matlab software. For the validation of the mathematical model, a DoE (23 with central point) was performed, where the sample load, velocity, and bed height were assessed as independent variables, whereas 99% purity and operating time were response variables. As the main results, simulated profiles did not show statistical differences (residual ≤ 5%) concerning experimental profiles. This study introduces a validated phenomenological model for SEC applied to purifying a TNF-blocking peptide, integrating computational simulations with experimental validation, enabling reliable process scale-up for industrial bioprocess design. In conclusion, it was possible to develop a phenomenological model of the SEC to ensure the purity of the antiTNF recombinant prototype at different scales, which allows the designing of industrial processes at scales of desired dimensions.