Boronic acid affinity media conjugating with PEG enable precise pH-responsive HPLC separation of glycoproteins depending on differences of sugar chains.

Boronic acids (BAs) reversibly bind to diol structures in a pH-dependent manner and have attracted attention as a method for separating glycoproteins based on their sugar chains. However, commonly used phenylboronic acid reacts only under basic conditions, which can cause protein denaturation and aggregation. To address this issue, we focused on BPA, BBX, PBA-CF3, and PBA-F, which are expected to have lower pKa values due to their electron-withdrawing groups. In this study, we designed and synthesized a novel stationary phase by immobilizing these BA derivatives onto silica particles for HPLC separation of glycoproteins. First, polyethylene glycol (PEG) was introduced as a spacer. Its high hydrophilicity and excluded volume effect suppressed non-specific protein adsorption. At the same time, glycoproteins such as horseradish peroxidase and immunoglobulin were retained even under neutral pH conditions, likely due to complex formation with BA derivatives based on differences in sugar chains. Next, polyethylenimine (PEI) was introduced to increase the immobilization amount of BA derivatives. As a result, the binding affinity toward glycoproteins increased, and a wider range of glycoproteins were strongly retained. Finally, HPLC with a pH gradient was performed, and the elution order of glycoproteins changed depending on the type of BA derivative. This indicates that glycoprotein separation can be controlled by both the BA derivative used and the mobile phase pH.