Activation of Supports Containing Hydroxyl Groups Using bis(4-Nitrophenyl) carbonate

Abstract

The hydroxyl groups carrying supports, Sepharose Cl-4B and Fractogel HW75F, were facilely activated under anhydrous conditions using bis(4-nitrophenyl) carbonate (BPNPC), a stable and readily available compound. The activation time at room temperature can be as short as 0.5 hour. The activated supports can be stored at 4°C for at least one month without any loss of coupling capacity. Amines can be coupled to the activated support at pH 7 to 10. Coupling of Protein A to BPNPC activated supports yielded immobilized Protein A affinity supports having a high IgG binding capacity. For example, the Protein A-Sepharose and Protein A-Fractogel prepared by using the BPNPC activation method were able to bind, respectively 13 - 14 mg and 8 - 9 mg human IgG with phosphate buffer saline as the loading buffer. In contrast the use of a high yield binding buffer increases the binding capacity of the two affinity supports to 25 - 28 mg and 22 mg respectively.

Key Words:

• bis(4-nitrophenyl) carbonate activated supports
• IgG purification
• affinity chromatography