Efficient capture of recombinant SARS-CoV-2 receptor-binding domain (RBD) with citrate-coated magnetic iron oxide nanoparticles.

Several vaccines against COVID-19 use a recombinant SARS-CoV-2 receptor-binding domain (RBD) as antigen, making the purification of this protein a key step in their production. In this work, citrate-coated magnetic iron oxide nanoparticles were evaluated as nano adsorbents in the first step (capture) of the purification of recombinant RBD. The nanoparticles were isolated through coprecipitation and subsequently coated with sodium citrate. The citrate-coated nanoparticles exhibited a diameter of 10 ± 2 nm, a hydrodynamic diameter of 160 ± 3 nm, and contained 1.9 wt% of citrate. The presence of citrate on the nanoparticles' surface was confirmed through FT-IR spectra and thermogravimetric analysis. The crystallite size (10.1 nm) and the lattice parameter (8.3646 Å) were determined by X-ray diffraction. In parallel, RBD-containing supernatant extracted from cell culture was exchanged through ultrafiltration and diafiltration into the adsorption buffer. The magnetic capture was then optimized using different concentrations of nanoparticles in the purified supernatant, and we found 40 mg mL-1 to be optimal. The ideal amount of nanoparticles was assessed by varying the RBD concentration in the supernatant (between 0.113 mg mL-1 and 0.98 mg mL-1), which resulted in good capture yields (between 83 ± 5% and 94 ± 4%). The improvement of RBD purity after desorption was demonstrated by SDS-PAGE and RP-HPLC. Furthermore, the magnetic capture was scaled up 100 times, and the desorption was subjected to chromatographic purifications. The obtained products recognized anti-RBD antibodies and bound the ACE2 receptor, proving their functionality after the developed procedure.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis.

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.