Preparation and characterization of 96-well microplates coated with molecularly imprinted polymer for determination and biosimilarity assessment of recombinant human erythropoietin.

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.

Preparation and characterization of pH-responsive polyacrylamide molecularly imprinted polymer: Application to isolation of recombinant and wild type human serum albumin from biological sources.

In this work pH-responsive neutral and cationic polyacrylamide molecularly imprinted polymers (nMIP and cMIP, respectively) were prepared for separation of recombinant and wild type human serum albumin (HSA, pI 4.7) using mixture of polymerization initiators. The effect of pH during preparation and adsorption stages at pI(HSA)±2.0 on binding capacity and selectivity; imprinting factor (IF) was thoroughly investigated. SE-HPLC and RP-HPLC were employed for thorough evaluation of the stability of HSA at the studied experimental conditions and for simultaneous determination of HSA and erythropoietin (EPO) in their mixtures, respectively. Results showed that nMIP were generally superior to cMIP, where nMIP prepared at pH 2.7 and tested at pH 6.7 showed superior binding characteristics (IF 42.91). The pH at the preparation stage imposed minimal effect on the stability of HSA owing to entrapment of HSA within the polymer network. Adsorption experiments carried out at pH 2.7, regardless of polymer type and pH of preparation revealed poor selectivity. Adsorption of HSA onto MIP followed Sips model with pseudo second-order kinetics. Scanning electron microscopy (SEM) revealed a rough surface for MIP and a smooth one with wider pore diameter for non-imprinted polymer (NIP). Successful separation of recombinant HSA from its binary mixture with EPO and wild type HSA from crude plasma was demonstrated using RP-HPLC. This suggested that MIP should be applicable for downstream purification of therapeutic grade HSA at scale either from plasma or recombinant sources and isolation of HSA from plasma for diagnostic purposes.