Innovative multifunctional tag system for protein purification and analytical characterization

Epitope tag-monoclonal antibody (mAb) systems are essential tools in biotechnology. We developed a novel system based on the mGMOP peptide-derived from GM-CSF with six O-glycosylation sites and an N-terminal APARSPS epitope-and the in-house mAb CC1H7, which binds this epitope, in single or multiple copies, with enhanced affinity under high ionic strength. The mGMOP-mAb CC1H7 system was applied to immunoaffinity chromatography, Western blot, and competitive ELISA. Optimization used two engineered interferon variants: one with a single tag (mGMOP-IFN) and another with four tags (mGMOP3-IFN-mGMOP). A third variant, mGMOP3-EPO-mGMOP, was constructed using erythropoietin for further validation. Immunoaffinity purification was optimized via factorial design, testing different salts. The final protocol used 1 M Na₂SO₄ for loading and 50 mM sodium phosphate at pH 11 for elution, achieving 80-92 % recovery and 100 % purity. Competitive ELISA was optimized using a Box-Behnken design, yielding low LOD and LOQ. Western blotting confirmed detection of both IFN variants, also with low LOD and LOQ. All methods were successfully applied to tagged EPO. The platform enables standardized workflows for characterizing recombinant proteins without requiring protein-specific antibodies, and offers a versatile approach for protein bioprocessing.