Development of Industrializable SMB Processes Based on New Configuration and Design Methods for Ultra-High-Purity Purifications of Oligonucleotide Monomers, Ribose, and Galactotriose

Abstract

Development of Industrializable SMB Processes Based on New Configuration and Design Methods for Ultra-High- Purity Purifications of Oligonucleotide-Drug Monomers, Ribose, and Galactotriose Cheol Yeon Jo Dept. of Chemical Engineering The Graduate School Hanyang University Recently, a simulated-moving-bed (SMB) technology has been acknowledged as one of the most reliable ways for establishing continuous-mode purification processes for the economical productions of valuable chemicals or biochemicals with high purities and high productivities. In this study, we aimed to develop the industrializable SMB processes that could be well-customized for highly-efficient purifications of three target products 2'-O-methoxyethyl (MOE) oligonucleotide monomer, ribose, and galactotriose), which have served as key building- blocks or raw materials of high-value pharmaceutical products in related industries. As a means of promoting such SMB process developmental works, we endeavored to devise a series of new SMB configurations, efficient SMB design strategies, and well-customized column arrangement modes that can lead to the best SMB performances. Particular attention was paid to ensuring that the performances of the developed SMB processes in this research could be maintained after scale-up, thereby accelerating its industrial application. To describe the aforementioned research work in detail, this paper was organized in a way to consist of three parts according to the target product. The first part was set to handle MOE oligonucleotide drug monomers, which is as follows. One of the commercially proven oligonucleotide-based drugs that can treat a variety of life-threatening diseases through gene- expression control is 2'-O-methoxyethyl (MOE) oligonucleotide. To keep the economical production of the MOE-oligonucleotide stable, it is important to establish an efficient process for recovering its corresponding monomer (MOE nucleoside phosphoramidites) in a large quantity at high purity from the output of the phosphitylation reaction based on using MOE nucleoside as a starting component. To address this issue, we pursued the development of an efficient simulated-moving-bed (SMB) process for continuous-mode recovery of monomer from the considered reaction output. First, the intrinsic parameters of monomer and impurities present in the reaction output were obtained through an inverse method using silica gel as an adsorbent. The resulting intrinsic parameters were then applied to the optimal design of the SMB process for monomer recovery, which revealed that its optimal configuration was a four-zone open-loop SMB configuration based on the installation of regeneration and re-equilibration zones for implementing a stepwise solvent-gradient method. Under such configuration, the optimal SMB operation parameters to maximize throughput while keeping high purity stable were determined using the relevant optimization tool based on column model and genetic algorithm. It was confirmed through the experimental run that the optimized SMB process could enable the continuous recovery of monomer from the considered reaction output with 99.6% purity while keeping the monomer loss close to zero. Finally, the developed SMB process in this article was found to increase throughput by more than 33 times, and reduce solvent usage by more than 89%, compared to the plant-scale batch chromatographic process currently in operation in related industries. The second part was set to handle ribose, which is as follows. The ultra-high-purity separation of ribose from the mixture of monosugars stemming from the hydrolysis of defatted microalgal biomass has been a key issue in the areas of microalgae-based ribose production process for pharmaceutical applications. To address such issue, this study was aimed at developing a customized simulated-moving-bed (SMB) process to steadily recover ribose with ultra-high purity from the aforementioned monosugar mixture. As a first step for this task, the intrinsic parameters of the mixture components containing ribose were determined through single-column experiments, which revealed the issue of a low selectivity between ribose and other impurities. On the basis of the determined intrinsic-parameters, the optimal design of the ribose-separation SMB was conducted in such a way that a proper degree of separation margin for maintaining an ultra-high level of ribose purity steadily throughout the SMB operation could be reflected in a quantitative manner while keeping the degree of the adsorbent-bed utilization as high as possible. These considerations were handled by carrying out the optimization runs of maximizing throughput while forcing the purity and yield constraints to be met not only in the normal situation (no deviations in adsorption parameters) but also in the situation where the adsorption parameters of all components would have a given range of fluctuations. The simulation and experimental works for such optimized ribose-SMB confirmed that it could lead to the continuous-mode separation of ribose with the purity of 99.6% without loss, and the degree of its adsorbent-bed utilization could be kept to the highest level when the worst cases of deviations in adsorption parameters would occur. The third part was set to handle galactotriose, which is as follows. The single-objective and multi-objective optimizations based on a multi- component standing-wave-design were carried out for the tandem simulated-moving-bed (SMB) process for continuous-mode separation of galactotriose (G3) from crude galacto-oligosaccharides. This process (named “G3-SMB”) consisted of two subordinate SMB units. The results from the G3-SMB optimization showed that its two subordinate SMB units had a significant difference in column efficiency, which acted as a major factor for restricting the level of attainable throughput and deteriorating solvent usage. This problem could be overcome by allowing the use of different column lengths within the G3-SMB (called “S1 method”), which was applied in a way that increased the column length of the subordinate SMB unit under the influence of column-efficiency limiting factor while decreasing the column length of the subordinate SMB unit with high column-efficiency. This method led to 32% increase in maximum achievable throughput and 37% reduction in solvent usage. It was also found that the adjustment of column configuration in each unit (called “S2 method”) was as effective in improving throughput as the S1 method, but less effective in reducing solvent usage than the S1 method. Finally, it was confirmed that the simultaneous use of the aforementioned S1 and S2 methods could create a synergy effect, thereby resulting in 74% increase in maximum achievable throughput and 39% reduction in solvent usage.