Optimization of protein entrapment in affinity microcolumns using hydrazide-activated silica and glycogen as a capping agent

Abstract

Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization that could be obtained by means of entrapment. The levels of retention and protein immobilization were evaluated by injecting warfarin or 4-methylumbellipheryl α-Dmannopyranoside as solutes with known binding properties for HSA or Con A. Altering both the solution volume and amount of added protein led to an increase of up to 17-fold in the extent of protein immobilization for HSA in slurry-based entrapment; on-column entrapment provided an additional 3.6-fold increase in protein content vs. the optimized slurry method. Similar general trends were seen for Con A. The protein contents obtained by entrapment for HSA or Con A (i.e., up to ~87 and 46 mg/g silica, respectively) were comparable to or higher than levels reported for the covalent immobilization of these proteins onto silica. The retention of warfarin on the entrapped HSA was at least 1.7-fold higher than has been obtained under comparable support and mobile phase conditions when using covalent immobilization. These results indicated that entrapment can be an attractive alternative to covalent immobilization for proteins such as HSA and Con A, with this approach serving as a potential means for obtaining good solute binding and retention in work with affinity microcolumns or related microscale devices.

Development of an on-line immunoextraction/entrapment system for protein capture and use in drug binding studies by high-performance affinity chromatography

“…Hydrazide-activated silica was prepared by using Nucleosil-300 silica as the starting material, which was first converted into a diolbonded form [8,19]. The diol-bonded silica was oxidized into an aldehyde form by using periodic acid, and hydrazide groups were incorporated by combining the aldehyde-activated silica with oxalic dihydrazide, as described previously [8,9]. Any remaining aldehyde groups on the support were reduced by using sodium borohydride [8,19].…”

“…Oxidation of glycogen was carried out by reacting 17 mg of bovine glycogen with 135 mg of periodic acid in 4.0 mL of pH 5.0, 20 mM acetate buffer containing 15 mM sodium chloride [9]. The glycogen solution was vortexed for 5 min, degassed for 5 min, and placed on a wrist shaker (set at 300 oscillations per min) for 16 h at room temperature.…”

“…column that contained hydrazide-activated silica. This step was done by first passing the captured protein repeatedly through the column for 4 h at 20 μL/min by using two syringe pumps that were synchronized to withdraw and infuse the protein solution through the column [9,12]. The HSA solution was then combined manually with an equal volume of a solution containing 4.25 mg/mL of oxidized glycogen; this mixture was circulated through the column for another 16-20 h. In the final step, 450 μL of pH 6.0, 0.10 M potassium phosphate buffer containing 1.0 mg/mL oxalic dihydrazide was applied for 2 h to the column to cap any remaining active aldehyde groups on the oxidized glycogen.…”

“…Protein entrapment by the method shown in Fig. 1 is normally performed at pH 5.0 to allow hydrazide groups on the support to react with oxidized glycogen and entrap a protein such as HSA [8][9][10][11][12]. However, other pH values that are above or below 5.0 may also be used for this process [19].…”

“…1) [8]. This method has previously been used to immobilize HSA and other serum proteins for use in HPAC and drug binding studies [8][9][10][11][12]. Advantages of this method include the ability of low-to-moderate mass drugs to access the entrapped protein and for protein to remain in a soluble and fully-active form [8,9].…”