Monolithic supports have become the subject of extensive study in the past years. Despite their advantageous features and many successful chromatographic applications in the analytical scale, only a very few examples of larger volume monoliths were described. In the case of GMA-EDMA monoliths, this can be attributed to the fact that due to the exothermic polymerization a pronounced temperature increase inside the monolith significantly affects the structure. The temperature increase depends on the thickness of the monolith, and consequently, there is an upper limit that allows the preparation of a unit with a uniform structure. In the present work, we have analyzed a heat release during the polymerization and have derived a mathematical model for the prediction of the maximal thickness of the monolithic annulus having a uniform structure. On the basis of the calculations, two annuluses of different diameters were polymerized and merged into a single monolithic unit with a volume of 80 mL. In addition, a special housing was designed to provide a uniform flow distribution in the radial direction over the entire monolith bed. It was shown that such a monolithic column exhibits flow-independent separation efficiency and dynamic binding capacity up to flow rates higher than 100 mL/min. The separation and loading times are in the range of a few minutes. The pressure drop on the column is linearly dependent on the flow rate and does not exceed 2.5 MPa at a flow rate of 250 ml/min.

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This review describes the novel chromatography stationary phase--a porous monolithic methacrylate-based polymer--in terms of the design of the columns and some of the features that make these columns attractive for the purification of large biomolecules. We first start with a brief summary of the characteristics of these large molecules (more precisely large proteins like immunoglobulins G and M, plasmid deoxyribonucleic acid (DNA), and viral particles), and a list of some of the problems that were encountered during the development of efficient purification processes. We then briefly describe the structure of the methacrylate-based monolith and emphasize the features which make them more than suitable for dealing with large entities. The highly efficient structure on a small scale can be transferred to a large scale without the need of making column modifications, and the various approaches of how this is accomplished are briefly presented in this paper. This is followed by presenting some of the examples from the bioprocess development schemes, where the implementation of the methacrylate-based monolithic columns has resulted in a very efficient and productive process. Following this, we move back to the analytical scale and demonstrate the efficiency of the monolithic column--where the mass transfer between the stationary and mobile phase is greatly enhanced--for the in-process and final control of the new therapeutics. The combination of an efficient structure and the appropriate hardware results in separations of proteins with residence time less than 0.1 s.

New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept--the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.