(249f) Continuous Recycling of an Immobilized Biocatalyst in a Solids Forming Reaction By Magnetic Separation

Enzymatic reactive crystallization is a promising green synthesis route for the production of semi-synthetic β-lactam antibiotics. End-to-end continuous manufacturing is one method to assist in careful control of the stable operating point that reactive crystallization requires. For β-lactam antibiotic synthesis, simulations have shown that use of continuous reactive crystallization can be optimized to increase productivity and conversion compared to batch synthesis [1]. One major limitation is in the use of a solid catalyst (immobilized enzyme) in the presence of crystallizing API and the resulting solid-solid separation that is required to purify the API and recycle the catalyst.

Solid-solid-liquid separations are difficult and uncommon design problems that are typical to the mineral industry, but rarely used in the pharmaceutical industry. One possible solution is the use of magnetic separation to separate enzyme immobilized to paramagnetic material from the nonmagnetic crystalline product. While magnetic separation has been implemented in several prior systems, including with the enzyme penicillin G acylase (PGA) as used in this study [2-4], the application detailed in this work is unique due to the continuous nature of the separation and its use at the pharmaceutical pilot plant scale.

In this study, a continuous magnetic separator was designed and optimized using iterative prototyping. Processing conditions that impact separation efficiency and solids throughput were evaluated using a Buckingham Pi analysis. Further optimization of the device was performed by separating the magnetic enzyme immobilization platform from nonmagnetic beads under flow rates from 3 – 15 mL/min. Separation efficiencies of >99% were obtained at flow rates and solid densities comparable to the current pilot plant used for cephalexin synthesis. The separator was then demonstrated in a batch operation for the separation of carrier and cephalexin crystals under conditions that mimicked the expected output from the pilot plant. Next, operation for the separator was demonstrated continuously for one and five hours, again with simulated pilot plant output conditions. Finally, the device was demonstrated continuously for 18 hours to separate and recycle immobilized PGA from cephalexin crystals generated from the enzymatic reactive crystallization of cephalexin at the pilot plant scale.

[1] McDonald, M.A., et al., Continuous reactive crystallization of β-lactam antibiotics catalyzed by penicillin G acylase. Part I: Model development. Computers & Chemical Engineering, 2019. 123: p. 331-343

[2] Luo, X., and Zhang, L. (2010) Immobilization of penicillin G acylase in epoxy-activated magnetic cellulose microspheres for improvement of biocatalytic stability and activities, Biomacromolecules 11, 2896-2903.

[3] Netto, C. G., Toma, H. E., and Andrade, L. H. (2013) Superparamagnetic nanoparticles as versatile carriers and supporting materials for enzymes, Journal of Molecular Catalysis B: Enzymatic 85, 71-92.

[4] Liu, R., Huang, W., Pan, S., Li, Y., Yu, L., and He, D. (2020) Covalent immobilization and characterization of penicillin G acylase on magnetic Fe2O3/Fe3O4 heterostructure nanoparticles prepared via a novel solution combustion and gel calcination process, International Journal of Biological Macromolecules.