PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.
60 mL of non-concentrated periplasmic fraction from Prevotella bryantii cultivation medium were subjected to two-step purification on CIM® DEAE 8 mL tube. Both purification steps were performed under similar chromatographic conditions, i.e. same column, buffer systems and flow rate (7 mL/min). Gradient slopes varied to achieve isolation of the desired fraction. The elution fraction of interest of purification No1 was subjected to a second purification on the same column. By altering the linear gradient pure 66 kDa-Endoxylanase for production of antibodies was isolated.
50 mL of crude enzyme solution were loaded on CIM® DEAE 8 mL tube and eluted by a linear gradient at a flow rate of 1.2 mL/min. Analysis of the elution fraction resulted in 86.1% recovery and 4 times concentration of the sample, having a purity of >95%.
A supernatant from Phanerochaete chrysosporium cultivation was loaded on CIM® QA Disk, and elution was effected by a linear gradient at a flow rate of 3 mL/min (9 CV/min). Baseline separation of isoenzymes H2, H6/H7, H8 and H10 was achieved in less than 3 minutes.