(369a) Enzymatic Reactive Crystallization of Cephalexin Using Immobilized Penicillin G Acylase from E. coli

Beta-lactam antibiotics have been used extensively to treat bacterial infections for nearly a century. The two largest produced beta-lactam antibiotic classes, semi-synthetic penicillins (e.g. amoxicillin) and cephalosporins (e.g. cephalexin), constitute the largest amount of sales of antibiotics for the treatment of bacterial infections. Currently the industrial production of beta-lactam antibiotics is conducted largely via batchwise chemical synthetic pathways consisting of multiple reaction steps, use of protecting groups, harsh solvents, and very low (-40°C) temperatures. Using these methods, the chemical synthesis route produces a large amount of chemical waste with an E factor of roughly 50 and is highly energy intensive due to the operation of low temperature refrigeration equipment. Additionally, due to increases in labor and raw material costs, much of the production of beta-lactam antibiotics has moved to China and India [1]. Considering recent events regarding COVID-19, the disruption international supply lines could lead to widespread shortages of highly important antibiotics in Western countries [2]. Improving the economic viability and environmental sustainability of beta-lactam antibiotic production is paramount to globalizing the production of these important drugs to decrease the chance of shortages.

Recently, we have been working to develop a continuous enzymatic beta-lactam antibiotic production process to address many of the issues of the current batchwise chemical synthesis processes to produce cephalexin and amoxicillin. These drugs were selected because they are the two highest volume manufactured and prescribed antibiotics worldwide and have been included in the world health organization (WHO) list of essential medicines as key access antibiotics [3]. Next, the move from chemical synthesis to enzymatic synthesis decreases waste production and increases yield due to high enantioselectivity of enzymes and the ability to operate in a mild temperature aqueous environment. We have expressed, purified, and engineered penicillin G acylase from E. coli (EcPGA), an industrially relevant enzyme used to produce precursors for semi-synthetic beta-lactam antibiotics, to synthesize cephalexin and amoxicillin. Additionally, we have immobilized EcPGA on various solid supports via covalent attachment, allowing for the recycle of the biocatalyst and decreasing the overall cost of the catalyst for production. Next, the induction of crystallization of the API within the reaction vessel protects the desired product from hydrolysis by EcPGA into a minimally soluble byproduct. The in-situ crystallization of the API enables the downstream separation of the solid crystals from the mother liquor into a filtration, washing, and drying protocol.

By combining these elements, we have conducted several successful seeded batch reactive crystallization experiments to produce solid cephalexin crystals. We found that after three crystallization experiments of three hours each, the catalyst suffered a 20% decrease in specific activity. This decrease may be due to crystallization within the pores of the solid catalyst, as the catalyst experienced no measurable deactivation under reaction conditions without crystallization. We have also studied the effect of different sized support particles as well as different support materials (e.g. agarose, methacrylate, polypropylene) on the diffusive properties of cephalexin and its precursors as well as on-line stability of the immobilized biocatalyst. Additionally, during reactive crystallization, after approximately four hours, the scarcely soluble byproduct, phenylglycine, generated enough supersaturation to precipitate and contaminate the solid phase. The progress of cephalexin crystallization and growth as well as phenylglycine precipitation were detected via in-situ microscopy and focused beam reflectance measurement (FBRM). We are currently working to implement these same elements and use these experiments to inform the design and operation of continuous MSMPR. The shift from batch to continuous production will improve the capacity and decrease the overall footprint of the production process as well as decrease the amount of off-specification product due to more robust control during steady state operation. The implementation of process analytical technology (PAT) such as Fourier transform infrared spectroscopy (FTIR) will allow for the on-line monitoring of the liquid phase components while FBRM and in-situ microscopy will allow for the on-line monitoring of the solid phase components. Additionally, reverse phase HPLC will be used to confirm final product purity. With the information of the components’ concentrations in both phases, we will study and develop a control strategy to maintain a “state-of-control” operation of the reactive crystallization process for the production of cephalexin and amoxicillin.

References:

[1] Elander, R. P. (2003). Industrial production of β-lactam antibiotics. Applied Microbiology and Biotechnology, 61(5-6), 385–392. doi: 10.1007/s00253-003-1274-y

[2] Oehler, R. L., & Gompf, S. G. (2020). Shortcomings in the US Pharmaceutical Supply Chain. Jama. doi: 10.1001/jama.2020.1634

[3] WHO Model Lists of Essential Medicines. (n.d.). The SAGE Encyclopedia of Pharmacology and Society. doi: 10.4135/9781483349985.n433