(18f) Improving Enzymatic Synthesis of ?-Lactam Antibiotics By in-Situ Crystallization
Matthew A. McDonald*, Lukas Bromig*, Andreas S. Bommarius*, Martha A. Grover*, and Ronald W. Rousseau*
*School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia
Keywords: reactive crystallization, Î²-lactam antibiotics, penicillin G acylase
In 2010 the two most common classes of Î²-lactam antibiotics, the penicillins and cephalosporins, accounted for nearly 60% of world antibiotic consumption, with delivery of over 20 billion doses of penicillin-derived antibiotics.1 Traditionally, Î²-lactam antibiotics have been made batch-wise in a process requiring several chemically intensive steps, such as amine protection by benzyl chloroformate and deprotection by hydrogenation with palladium catalysts, resulting in large amounts of potentially contaminated waste.2 The contaminated waste poses two threats: the spread of antibiotic resistance in wastewater treatment plants3 and inadvertent exposure of sensitive individuals to a sever allergen. A 2012 study found that 92 out of 11,761 patients at Mount Sinai Hospital in New York City suffered anaphylaxis (a rapid, potentially deadly allergic reaction) from penicillin, a prevalence of 0.7%.4Enzymatic synthesis of Î²-lactam antibiotics can help to mitigate these risks by operating in mild aqueous conditions. The enzyme used industrially for this purpose is penicillin G acylase (PGA); it also catalyzes the hydrolytic degradation of the antibiotic (termed secondary hydrolysis), making the desired product an intermediate.Â In-situ product isolation can prevent the enzyme from degrading the antibiotic.
We use ampicillin and cephalexin as a model penicillin and cephalosporin, respectively. PGA catalyzes the condensation of 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester (PGME) into ampicillin and 7-aminodesacetoxy-cephalosporanic acid (7-ADCA) and PGME into cephalexin. Ampicillin and cephalexin have lower saturation concentrations than 6-APA and 7-ADCA, respectively, making it possible to supersaturate the solution with respect to the antibiotic. The supersaturation can then be used to crystallize pure ampicillin and cephalexin, isolating it from PGA.5Degradation of ampicillin and cephalexin by PGA yields phenylglycine, a sparingly soluble contaminant. PGA also hydrolyzes the highly soluble PGME into phenylglycine (termed primary hydrolysis). To maintain a pure solid phase phenylglycine must not be allowed to reach its saturation concentration. Typical crystallization processes utilize evaporation, cooling, and other chemical process to generate supersaturation. In this process, PGA generates the supersaturation necessary for the separating phase change (from solute to crystal).
To better understand the mechanism and quantitatively predict the process behavior, a model of the enzyme kinetics, including the synthesis, primary, and secondary hydrolysis reactions, was developed. Existing kinetic models for ampicillin and cephalexin synthesis by PGA do not consider the effects of pH value on the reaction despite the fact that pH is known to affect the enzyme selectivity and activity. The pH value is shown to decrease with increasing conversion because of the generation of an acid group in phenylglycine. We have improved on previous models such that our model considers the effect of pH and removal of species from solution via crystallization.6
For ampicillin the new process showed maximum improvements in yield and selectivity of 33% and 49% over the non-crystallizing control, respectively, all while maintaining a strict purity constraint. However, these two maxima cannot be simultaneously achieved; instead selectivity is optimized at a lower pH value around 6 where the enzyme is suspected to bind 6-APA more strongly, and yield improves at a higher pH value around 7 where catalytic activity is improved but less specific.6Preliminary results show cephalexin enjoys similar benefits from reactive crystallization.
More recently we have explored continuous reactive crystallization to synthesize Î²-lactams because it may enable greater efficiency and access to different chemistries. To operate a continuous process the stability and longevity of the enzyme must be characterized. We have determined the temperature activity profile and the empirical parameter T6050, the temperature at which 50% of the initial activity degrades within one hour, of an industrially relevant PGA, AssemblaseÂ® from DSM-Sinochem. . Temperature ramp studies were also used to determine catalyst longevity (in the form of total turnover number, TTN). Continuous operation could increase conversion over batch by tailoring the conditions to optimize reactive crystallization; for example, a plug flow reactor/crystallizer can operate with excess 6-APA or 7-ADCA relative to PGME to hinder hydrolysis of the PGME while a recycle stream could act to seed the plug flow apparatus to slow antibiotic hydrolysis via crystallization. The aforementioned model is combined with enzyme stability data to map the large design space opened by continuous operation.
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 Giordano, R. C., Ribeiro, M. P., and Giordano, R. L. (2006) Kinetics of Î²-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization, Biotechnology advances 24, 27-41.
 Deschamps, E., Vasconcelos, O., Lange, L., Donnici, C. L., Silva, M. C. d., and Sales, J. A. (2012) Management of effluents and waste from pharmaceutical industry in Minas Gerais, Brazil, Brazilian Journal of Pharmaceutical Sciences 48, 727-736.
 Albin, S., and Agarwal, S. Prevalence and characteristics of reported penicillin allergy in an urban outpatient adult population, 6 ed., pp 489-494, OceanSide Publications, Inc.
 EncarnaciÃ³n-GÃ³mez, L. G., Bommarius, A. S., and Rousseau, R. W. (2016) Reactive crystallization of Î²-lactam antibiotics: strategies to enhance productivity and purity of ampicillin, Reaction Chemistry & Engineering 1, 321-329.
 McDonald, M. A., Bommarius, A. S., and Rousseau, R. W. (2017) Enzymatic reactive crystallization for improving ampicillin synthesis, Chemical Engineering Science 165, 81-88.