Electrokinetic assessment of RNase A species and innovative PEG-grafted agarose-based resins used in downstream processing of PEGylated proteins

Abstract

Detailed study of the governing mechanisms of hydrophobic interaction chromatography in protein purification plays a key role in the design of separation systems. In our previous work, an innovative covalently bonded poly(ethylene glycol) (PEG) to Sepharose® 6B resin was developed. It could successfully separate PEGylated ribonuclease A (RNase A) species in a single chromatographic step (Hernández-Martínez and Aguilar, 2014), however, its electrokinetic profile has not been evaluated. The aim of this work was to study electrokinetics of RNase A species, unactivated, activated and activated-PEGylated Sepharose® 6B resins, and 5–40 kDa PEGylated resins. Electric potential, mobility and conductivity of resins was determined in presence of 0.025 M potassium phosphate buffer pH 7.0 supplemented with ammonium sulfate. Scanning electron microscopy and infrared spectroscopy techniques were used to characterize the resins. All protein and resins surfaces were negatively charged at pH 7.0 and ζ potential was dependent on electrolyte ion concentration. Variation in PEG molecular weight caused variation in ζ potential at low electrolyte concentrations. Morphology of resins did not change after resin PEGylation, however, size of particles ranged between 8–55 μm. These results contribute to electrokinetics understanding of RNase A and PEG-grafted resins, providing electric characterization of their surfaces, which is essential in the design, improvement and problem-solving in chromatographic methods for PEGylated species purification.