(601d) Islatravir Penultimate Step Crystallization: Purification of an API in the Presence of High Protein Loading from an Enzymatic Cascade

Margelefsky, E. - Presenter, Merck & Co., Inc.
Robaire, S. A., Merck & Co., Inc.
Kwok, T. T., Georgia Institute of Technology
Miller, M., Merck & Co., Inc.
Heltzel, J., George Washington University
Sirk, K., Merck
Mattern, K. A., Merck & Co. Inc.
Forstater, J. H., Merck & Co. Inc.
Grosser, S. T., Merck & Co. Inc.
Aaron, W., Merck & Co., Inc.
Hughes, G., Merck & Co. Inc.
Mohan, A., Merck & Co.
Islatravir is a novel nucleoside active pharmaceutical ingredient (API) currently in clinical trials for both HIV treatment and prevention. As reported recently in Science (Huffman et. al. 2019), the API synthesis under development involves a 9-enzyme 3-step in-vitro cascade without intermediate isolations until the final API molecule is assembled. This process represents an incredibly efficient synthesis of the API molecule, but introduces significant challenges to the downstream purification of the product. Enzymes, host-cell proteins, bacterial endotoxins, residual nucleic acids, and organic impurities/related substances all must be removed to generate a drug substance of adequate purity for human dosing.

The primary challenge in the purification development has been on enzyme/protein removal. The presence of high levels of protein tends to make filtrations very slow, so extractive work-ups are often preferable, but the relatively low solubility of the API in most organic solvents was a formidable obstacle to this approach. Several extractive work-ups were developed but each posed stability concerns.

Additionally, enzyme immobilization was pursued for the enzymes with the highest loadings in the reaction, which improved downstream processing but added a significant cost and supply-chain complexity.

In the final reaction, the API crystallizes from the reaction mixture, and even wth several enzymes removed via immobilization, filtration was still very challenging. Filtration was optimized by adding filter aid (diatomaceous earth), reducing shear in the reaction, adding seed crystals to the reaction, and agitating the batch intermittently during filtration. Optimizing the filtration step to isolate the crude API made it possible to produce quality API without the need to develop an extractive work-up or use immobilized enzymes.


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