(524c) Optimizing Reverse Phase Chromatography Separation in Molnupiravir Synthesis: An Inverse Method Approach

The COVID-19 pandemic has resulted in millions of confirmed cases and deaths, placing an enormous burden on global healthcare systems. Molnupiravir, a potent antiviral agent that inhibits the replication of SARS-CoV-2, has been granted Emergency Use Authorization (EUA) status in the United States.¹ The production of molnupiravir involves the synthesis of its key intermediates, the 5'-isobutyrate ester and hydroxylamine, and their subsequent separation to prevent contamination.²

Reverse phase chromatography (RPC) is a widely used technique in the biopharmaceutical industry to separate non-polar and moderately polar compounds based on their hydrophobicity. However, determining kinetic parameters for nonlinear separations and optimizing operating conditions for RPC columns can be challenging. In this study, we utilized the inverse method to determine the isotherm constants for the adsorption of hydroxylamine and isobutyrate products onto an aqueous isocratic solution of acetonitrile and formic acid, providing a time- and resource-efficient approach.^{3, 4} The chromatographic separation investigated under this study is part of continuous manufacturing of drug substances (API).

In this study, we investigated the effects of several key parameters on the performance of reverse phase chromatography (RPC) for the separation of the 5'-isobutyrate ester and the hydroxylamine intermediate. Specifically, we examined the effects of flow rate, column geometry, dispersivity coefficient, and injection volume on the retention time, peak shape, peak width, and separation efficiency of chromatographic peaks. Our findings demonstrate that these parameters play a significant role in determining the quality of the separation. We observed that slower flow rates and longer column lengths led to broader peaks and longer retention times, while larger column diameters resulted in slower linear velocity of the mobile phase and extended retention times. Furthermore, a higher dispersivity coefficient and injection volume resulted in solute bands expanding and interacting more with the stationary phase, ultimately altering the shape and retention time of the peak. Overall, our study highlights the importance of optimizing these parameters to achieve efficient chromatographic separation, which is crucial for the successful production of molnupiravir and the development of effective antiviral drugs for COVID-19. The developed mechanistic model and this study could be also adapted to manufacture the drugs incase of future pandemic with less time and resources.

The insights gained from our study can provide valuable guidance for future research in the pharmaceutical and biopharmaceutical industry on the optimization of chromatographic separation. By understanding the effects of column geometry and operating conditions on the separation performance of chromatography, we can design more efficient processes for separating similar compounds. Ultimately, this will lead to the production of high-quality pharmaceutical products at a lower cost and in a more sustainable manner.

Acknowledgement:

This work is supported by the US Food and Drug Administration (FDA) under contract number 75F40121C00106.

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