Integrated Computational and Experimental Analysis of Recombinant Trastuzumab in ExpiCHO-S™ Cells

In this work, a computational and experimental methodology for the design, expression, purification, and first biochemical characterization of recombinant trastuzumab is described. HADDOCK was used for molecular docking in order to examine the interaction between trastuzumab and the HER2 target. The anticipated binding orientation, interface interactions, and energetic contributions within the antibody-antigen complex were all revealed by the docking analysis. Stable complex formation was indicated by the top-ranked docking clusters' constant electrostatic and van der Waals contacts, good HADDOCK scores, and low intermolecular RMSD values. These results support the formation and propagation of a mammalian expression construct expressing trastuzumab in Escherichia coli. Plasmid isolation and colony screening were used to find positive transformants. The verified construct was subsequently transiently transfected into ExpiCHO-S™ cells for antibody expression, with maintained cell viability reported during the expression period. Protein A affinity chromatography was used to purify secreted trastuzumab, and size-exclusion high-performance liquid chromatography (SEC-HPLC) was used to evaluate its purity profile. A prominent monomeric signal with little discernible aggregation was shown by the chromatographic analysis. SDS-PAGE was used to further investigate the fractions that corresponded to the primary SEC peak in both reducing and non-reducing conditions. Bands compatible with intact IgG and the anticipated heavy and light chains following reduction were shown by electrophoretic analysis. Overall, the study shows that it is possible to produce and assess recombinant trastuzumab by combining computational modeling with mammalian expression and biochemical characterization techniques.