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Abstract: Synthesis and applications of molecularly imprinted polymers (MIP) are rapidly growing. In this study, a biomimetic MIP was prepared through silanes polymerization on the surface of 96-well microplates using recombinant human erythropoietin-alfa (rhEPO) as a template molecule. The rhEPO was immobilized onto the plate surface using bi-functional cross-linker and a thin imprinted layer following sol-gel procedure was constructed. After template extraction, uniform three-dimensional cavities compatible with the configuration of rhEPO were obtained. The rhEPO-MIP preparation was optimized using 2-level factorial design and response surface design where polymerization time and interactions between the different variable were found to be the most significant factors. Size-exclusion chromatography (SEC) was used to monitor the stability of the rhEPO under the investigated polymerization conditions. Determination of rhEPO using the MIP microplate showed good dynamic response fitting to the 4 PL regression model (0.9962) over a concentration range of 10.00 - 100.00 ng mL-1. Adsorption of rhEPO onto MIP followed the Langmuir isotherm model (r = 0.9957, χ2 =0.02786) with pseudo-second-order kinetics (r = 0.9984). The surface of the rhEPO-MIP was characterized using scanning electron microscopy (SEM) while step-by-step surface modification was tracked using Fourier transform infrared (FTIR) spectroscopy. The rhEPO-MIP was able to distinguish between the rhEPO-alfa template and modified rhEPO molecules; rhEPO-beta, hyperglycosylated and pegylated forms (imprinting factors < 2) and in the commonly used formulation additive human serum albumin (HSA) (R% = 113.96 -95.22%). The rhEPO-MIP was applied to compare the receptor-binding pattern to rhEPO and its biosimilars / structural analogues. The results were cross-validated using the conventional assay protocol (RP-HPLC and ELISA) and an acceptable correlation was observed with RP-HPLC (maximum deviation is 7.78%). This work confirmed the applicability of rhEPO-MIP with its unique binding features for batch release, stability and biosimilarity assessment as well as subsequent evaluation of batch-to-batch consistency during bioproduction of target analytes
Template and target information: human erythropoietin-alfa, rhEPO
Author keywords: Recombinant human erythropoietin, Imprinted microplate, Template immobilization, Factorial design, Response surface design, artificial antibody