(410b) Combining Reaction with Crystallization and a Size-Based Biocatalyst Separation for the Continuous Manufacturing of Amoxicillin

Amoxicillin is a highly important drug used in the treatment of bacterial infections, and the largest produced antibiotic by volume globally. Its manufacturing has traditionally been conducted in a batchwise chemical synthetic process consisting of multiple steps, cryogenic refrigeration, along with the use of protecting groups and hazardous solvents. Research has been conducted in investigating the use of biocatalytic processing routes, which consist of a single reaction step, and are generally conducted in mild, aqueous conditions, generating much less waste and resulting in a fewer processing steps[1]. On the other hand, the biocatalyst, generally penicillin G acylase (PGA) from E. coli, not only catalyzes the one-step synthesis reaction, but also the hydrolysis of both the activated side-chain donor 4-hydroxyphenylglycine methyl ester (4-HPGME) as well as the product amoxicillin. Several strategies have been investigated to limit these side reactions in similar drugs, such as in situ product removal via complexation[2], aqueous two-phase systems[3], supersaturation of substrates[4], and more. While these strategies offer improvements over a standard one-pot aqueous system, they either increase difficulty in downstream processing steps or are only applicable to batch systems. Nevertheless, many improvements to the biocatalytic processing of amoxicillin are still required to make it feasible on an industrial scale.

In this work, we combine the reaction and crystallization in the same mixed suspension mixed product removal (MSMPR) vessel to improve the selectivity and productivity of the system as well as intensify the overall process. Additionally, we operate the process in a continuous fashion, leading to increased productivity, decreased process footprint, and easier operation in comparison to a batch process. Biocatalyst retention within the reactor was achieved by immobilizing PGA on a solid support of a defined size range (300-425 μm), and withdrawal of the product slurry through a stainless-steel mesh filter of 300 μm, thereby achieving a size-based separation between product crystals and biocatalyst. As amoxicillin trihydrate crystals will grow much larger than 300 μm normally without intervention, an external wet-mill loop was employed to decrease their size below the mesh filter threshold. Additionally, crystallization of amoxicillin was observed to be rate limiting, so we investigated the use of a second MSMPR, operated in the absence of biocatalyst, to allow for its complete desupersaturation from solution. It was determined that a residence time of 1 hour was sufficient to desupersaturate amoxicillin and improve product yield by 10%. Lastly, while these process improvements led to a high productivity of 500 g/L/d, the conversion of the beta-lactam donating moiety, 6-aminopenicillinoic acid (6-APA), and the side-chain donor, 4-HPGME, were low at only 53% and 40%, respectively. To address this limitation, we were able to isolate both substrates separately via pH-swing crystallization at pH values of 4.0 and 8.0 for 6-APA and 4-HPGME, respectively, exploiting their solubility’s opposite sensitivity to changes in pH. With the recovery and reuse of substrates, the overall conversion, defined as the fraction of the compound fed into the system which does not leave the system as waste, was improved to greater than 90% for both substrates.

1. McDonald, M.A., A.S. Bommarius, and R.W. Rousseau, Enzymatic reactive crystallization for improving ampicillin synthesis. Chemical Engineering Science, 2017. 165: p. 81-88.
2. Li, D., et al., Enhanced enzymatic production of cephalexin at high substrate concentration with in situ product removal by complexation. Food Technology and Biotechnology, 2008. 46: p. 461+.
3. Aguirre, C., et al., Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems. Process Biochemistry, 2010. 45(7): p. 1163-1167.
4. Youshko, M.I., et al., Penicillin acylase-catalyzed synthesis of ampicillin in “aqueous solution–precipitate” systems. High substrate concentration and supersaturation effect. Journal of Molecular Catalysis B: Enzymatic, 2000. 10(5): p. 509-515.