(430a) A New Method for the Synthesis of Gigaporous Polymer Beads

Liquid chromatography has been a center-stage technology for the purification of biomolecules in modern-day biotechnology. Various stationary-phase media have been developed in the last century for liquid chromatography. They include rigid inorganic particles (such as silica beads), soft polymeric gel particles (such as agarose beads), and rigid polymeric particles. Rigid polymer resins possess a higher mechanical strength than soft gels, and they provide a wider applicable pH range (between 1 - 12) than inorganic supports such as silica beads that are unstable at alkaline pH. Cross-linked polystyrene resins are the most popular rigid polymeric particles.

In recent years, the biomolecular products from the biotechnology industry present new challenges for liquid chromatography. Because some of the biomolecules have very large complex structures and are fragile, it is desirable that purifications be conducted as quickly as possible. Due to their large molecular sizes, some biomolecules require the polymeric particles to have very large macropore sizes. Most polymeric particles, however, contain macropores with a pore size range of 100 - 300 angstrom resulting in a longer separation time for the biomolecules due to slow diffusion of these molecules through the interior of the stationary-phase particles. Some polymeric particles are not spherical resulting in a less desirable column packing structure. In order to facilitate mass transfer and to make binding sites more accessible to large biomolecules, spherical polymer particles with very large pores permitting convective flow are desired.

A new method for the synthesis of polymer beads with very large macropores was developed recently. These beads were prepared by a suspension polymerization method. An oil phase was prepared by mixing 5.0 g styrene monomer, 5.0 g crosslinking agent (divinylbenzene), 0.40 g initiator, 0.5g diluent and a key component that played a key role in the formation of large pores in a reaction vessel. A water phase was prepared by dissolving a suspension agent (polyvinyl alcohol), a surfactant and an inhibitor in 100 ml distilled water. Then, an emulsion was prepared by dispersing the oil mixture into the water phase at a stirring rate of 150 rpm. After sparging the reaction mixture with nitrogen gas, its temperature was raised to 75^oC. The polymerization was carried out under a nitrogen atmosphere for 20 h at a stirring rate of 160 rpm. The polymer beads were filtrated and washed with water and then with methyl alcohol for several times. The beads were vacuum dried for one day. SEM was used to characterize the polymer beads. The beads produced using an optimized recipe were found to have a pore surface area of 204 m²/g, porosity of 83.6% and the nominal pore size is around 5000 angstroms. The particle diameter was in the range of 50 - 100 micron.

Because the polymer synthesis was conducted in a single reaction step, this new method is different from that used for the POROS perfusion chromatography media that require two separate reaction steps with the first producing small sub-particles before an assembly in a subsequent reaction. The effects of the crosslinking degree, the amounts of diluent and the key component, and the water phase composition on the structure of new polymer beads were investigated. Pore sizes were controlled by adjusting the crosslinking degree, the amounts of the key component and diluent. It was found the pore sizes decreased when the crosslinking degree increased. There were two sets of pores in the particles, one big and one small. The pore sizes and the number of bigger pores increased with the increase of the amount of the key component. The pore sizes and the number of smaller pores increased with the increase of the amount of the diluent. The particle sizes were controlled by the stirring speed and the water phase composition.

The surface chemistry of the new polymer beads can be readily modified for ligand attachments. Many chromatographic media such as affinity, size exclusion, adsorption and ion-exchange media can be produced using the new polymer beads.  In addition to chromatography applications, the macroporous polymer beads can be used as microcarriers in fungal and animal cell cultures as well as in drug delivery. They provide sufficiently large surface areas through their readily accessible large pore while still retaining a good physical strength.