Dielectrophoretic behavior of PEGylated RNase A inside a microchannel with diamond‐shaped insulating posts

Ribonuclease A (RNase A) has proven potential as a therapeutic agent, especially in its PEGylated form. Grafting of PEG molecules to this protein yields mono‐PEGylated (mono‐PEG) and di‐PEGylated (di‐PEG) RNase A conjugates, and the unreacted protein. Mono‐PEG RNase A is of great interest. The use of electrokinetic forces in microdevices represents a novel alternative to chromatographic methods to separate this specie. This work describes the dielectrophoretic behavior of the main protein products of the RNase A PEGylation inside a microchannel with insulators under direct current electric fields. This approach represents the first step in route to design micro‐bioprocesses to separate PEGylated RNase A from unreacted native protein. The three proteins exhibited different dielectrophoretic behaviors. All of them experienced a marked streaming pattern at 3000 V consistent with positive dielectrophoresis. Native protein was not captured at any of the conditions tested, while mono‐PEG RNase A and di‐PEG RNase A were captured presumably due to positive dielectrophoresis at 4000 and 2500 V, respectively. Concentration of mono‐PEG RNase A with a maximal enrichment efficiency of ≈9.6 times the feed concentration was achieved in few seconds. These findings open the possibility of designing novel devices for rapid separation, concentration, and recovery of PEGylated RNase A in a one‐step operation.