Design of Continuous Enzymatic Reactive Crystallization for Beta-Lactam Antibiotic Synthesis

Design of Continuous Enzymatic Reactive Crystallization for Beta-lactam Antibiotic Synthesis

Matthew A. McDonald¹, Andreas S. Bommarius¹, and Ronald W. Rousseau¹

¹School of Chemical and Biomolecular Engineering,

Georgia Institute of Technology, Atlanta, GA 30332-0100, USA

                  β-lactam antibiotics, despite a long and storied history, find continued widespread use in our fight against infection.  In 2010 the two most common classes of β-lactam antibiotics, those derived from penicillin and cephalosporin, accounted for nearly 60% of world antibiotic consumption, with over 20 billion doses given of penicillin-derived antibiotics alone [1].  Their high rate of use, both historical and contemporary, has contributed to a growing deadly allergy to β-lactams, particularly those derived from penicillin.  The rate of allergy is significant enough for the FDA to recently require that all penicillin-derived antibiotics be manufactured, processed, and packaged in facilities separate from those used for any other pharmaceuticals [2].  Traditionally, β-lactam antibiotics have been made through chemical synthesis requiring use of several protection groups and resulting in unsustainable amounts of potentially contaminated waste [3]. 

                  An alternative route that has received considerable interest involves using the enzyme penicillin G acylase (PGA) to synthesize semi-synthetic penicillin-derived antibiotics from an acyl donor (e.g. phenylglycine methyl ester for ampicillin) and 6-aminopenicillanic acid (6-APA) [3].  However batch processing, even enzymatic, results in only moderate yields and thus inefficient use of materials.  PGA also catalyzes the hydrolysis of the acyl donor (primary hydrolysis) and the hydrolysis of the antibiotic (secondary hydrolysis, ampicillin cleaves into phenylglycine and 6-APA), leading to unnecessary waste. 

                  This work focuses on the design of continuous reactive crystallization of ampicillin catalyzed by PGA, such that the precipitated ampicillin is protected from secondary hydrolysis and is of high purity.  The design of such a system requires models for kinetics of reaction and crystallization, solubility of all components, and robust controls for continuous operation.  The dependence of the kinetics and solubility on pH promises a means of control in which yield and selectivity can be maximized.

                  Crystallization kinetics (nucleation and growth) were determined via online chord-length distribution and concentration versus time data in batch crystallizers [4].  Change in pH was used to induce supersaturation, and the effect of crystallization on pH was recorded.  Since the goal is steady state operation of a continuous reactive crystallizer, seed crystals should be used in all experiments, the loading of seed crystals being another important design parameter.  Crystallization kinetics were combined with the reaction kinetics scheme of Youshko and Svedas [5].  The reaction kinetics were modified to include the effect of enzyme protonation state on the rates of synthesis and primary and secondary hydrolysis.  Batch experiments across a range of pH values were used to factor in the equilibrium of the enzyme-substrate complex and enzyme-product complex protonation states.  The effect of pH on the rates of ampicillin synthesis, primary hydrolysis, and secondary hydrolysis were determined independent of each other.  A combined model was used to determine the yield and selectivity for ampicillin over the hydrolysis product.  The largest obstacle to high conversion remains the accumulation and precipitation of the hydrolysis product phenylglycine, an intolerable impurity in the final project.

                  Model simulations show that continuous operation can increase yield considerably over batch processes.  Simulations also show the substantial benefit of crystallization and reaction occurring in the same vessel.  Different vessel designs (i.e. stirred tank, tubular reactor) have also been investigated via the model and show significant differences between competing designs.  A continuous stirred tank arrangement will be implemented experimentally to validate the model.  Preliminary results also suggest controls over important variables including crystal size distribution are possible while maintaining high yield, selectivity, and purity.

[1] Gelbrand, Helen, et al. "The State of the WorldÕs Antibiotics 2015." Wound Healing Southern Africa 8.2 (2015): 30-34.

[2] 21 C.F.R ¤ 211.42 2015

[3] Giordano, Roberto C., Marcelo PA Ribeiro, and Raquel LC Giordano. "Kinetics of β-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization."Biotechnology advances 24.1 (2006): 27-41.

[4] Encarnación-Gómez, Luis G., Andreas S. Bommarius, and Ronald W. Rousseau. "Crystallization Kinetics of Ampicillin Using Online Monitoring Tools and Robust Parameter Estimation." Industrial & Engineering Chemistry Research 55.7 (2016): 2153-2162.

[5] Youshko, M. I., and V. K. Śvedas. "Kinetics of ampicillin synthesis catalyzed by penicillin acylase from E. coli in homogeneous and heterogeneous systems. Quantitative characterization of nucleophile reactivity and mathematical modeling of the process." Biochemistry (Moscow) 65.12 (2000): 1367-1375.