Lyophilization of CNBr-activated agarose beads with lactose and PEG

CNBr-activated agarose beads (CAABs) have been considered as an important intermediate to derivate various versatile bio-separation media. Unfortunately, the highly reactive groups on the beads are liable to hydrolysis and therefore lyophilization is needed for long-term preservation. In the present study, it was found that although reactive groups could be protected with Na2HPO4–citric acid buffer (20 mM, pH 4.0), the spherical shape of the CAABs, which correlate to purification performance and flow rate in the separation application, could not be recovered after rehydration. Such permanent loss of spherical shape was attributed to the interchain rearrangement between originally distant reactive group and hydroxyl group of CAABs when collapse happened during lyophilization. Lactose and PEG were used as additives afterwards to obstruct the interchain rearrangement. Results showed that 10% PEG8000 and 10% (w/v) lactose in Na2HPO4–citric acid buffer (20 mM, pH 4.0) maintained the spherical shape well. Characterization of lyophilized CAABs revealed that the developed method could keep CAABs’ original properties well and the lyophilized CAABs can be used in preparing affinity chromatographic media to purify protein.