(179d) Dielectrophoretic Behavior of Pegylated Ribonuclease a in a Microdevice with Diamond-Shaped Insulating Posts

Ribonuclease A (RNase A) has proven potential applications as a therapeutic drug for tumor treatment, especially in its PEGylated form. Grafting of polyethylenglycol (PEG) molecules to this protein yields mono-PEGylated (mono-PEG) and di-PEGylated (di-PEG) RNase A conjugates and the remaining unreacted protein after reaction. From these species, mono-PEG RNase A has the highest biological activity. Recovery of mono-PEGylated RNAse A is a challenging task. Unlike chromatographic methods, which have been traditionally used to separate and recover mono-PEG RNase A, the use of electrokinetic foreces in microdevices represents an attractive option for this purpose. This work describes the dielectrophoretic behavior of PEGylated RNase A species and its native form inside an insulator-based dielectrophoresis (iDEP) device in route to design a microbioprocess to recover mono-PEGylated RNase A from the whole PEGylation mixture. The 10 mm long microdevice has a single row of six diamond-shaped posts embedded along the center of channel, each post having a half diamond placed on both sidewalls resulting in a device with two openings. The geometric array allowed the creation of high non-uniform electric fields when direct current electric fields were generated. As expected, the three proteins under study (i.e. native, mono-PEG, and di-PEG) exhibited different and distinct dielectrphoretic behaviors. Strong trapping of mono-PEGylated RNase A was observed when an electric potential of 4000 V was applied (Figure 1A). On the other hand, di-PEGylated RNase A showed a similar response when the electric potential applied was of 2500 V (Figure 1B). In the case of the native RNase A, no trapping was observed. The distinct dielectrophoretic behaviors of the three proteins open the possibility of designing novel devices to separate, concentrate, and recover one single PEGylated conjugate in a one step operation minimizing time, sample size, and reagent usage. This technique can serve as an alternative to traditional chromatographic methods in route to bioprocess miniaturization.