Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: Unfolded proteins and PEGylated proteins

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

“…In this study we focused on electrostatic interaction-based separation, i.e., IEC.The elution order is basically similar to that observed by Seely and Richey [6] and others [9,[11][12][13].…”

“…Using activated PEG molecules of different molecular weights, lysozyme was randomly PEGylated and BSA mono-PEGylated. Linear gradient elution (LGE) experiments were carried out, and the number of binding sites was determined [13][14][15] to examine how PEGylation affects the binding mechanism. For lysozyme, enzyme activity was measured using two different substrates (suspended or soluble) to determine how the PEG chain affects the enzyme activity.…”

“…The reaction mixture was kept in a thermostated shaker at 277K for 24-48 h. The final PEG -to-lysozyme molar ratio was 6 [13].…”

“…4 and 5). The number of binding sites, B values and the parameter A (including the equilibrium coefficient, the binding site and the ion-exchange capacity) were determined from the GH-I R curves [13][14][15] (Tables 1 and 2). This method is very suitable for this type of difficult separation as a number of peaks must be separated, which is not easy by a standard isocratic elution method.…”

“…PEGylated proteins can be separated by various types of chromatography, e.g., size exclusion chromatography (SEC), hydrophobic interaction chromatography and electrostatic interaction chromatography (ion-exchange chromatography, IEC) [5][6][7][8][9][10][11][12][13]. However, the separation mechanism is still not fully understood.…”