Crystallization Kinetics of Cephalexin Monohydrate in the Presence of Cephalexin Precursors

Abstract

The solubility and crystallization kinetics of cephalexin monohydrate, an important β-lactam antibiotic, are reported for the first time for an aqueous solution. The nucleation and growth kinetics are determined with online process analytical technologies (PAT) and offline image analysis to support implementation of a continuous process. Effects of two cosolutes, 7-aminodesacetoxycephalosporanic acid (7-ADCA) and phenylglycine methyl ester (PGME), on nucleation and growth are examined. These cosolutes are precursors in the enzymatic synthesis of cephalexin. Primary nucleation of cephalexin was unaffected by the cosolutes, but 7-ADCA, even in small quantities (<10 mM), significantly decreased the cephalexin monohydrate growth rate; PGME had a less significant impact on growth rate. Possible mechanisms of growth inhibition are discussed; a model wherein cosolutes slow growth by pinning advancing growth steps and halting step advancement below a critical supersaturation best explains the data. Impact on crystal purity is also discussed. The needle-like shape of cephalexin monohydrate caused two mixing issues: (1) at low stir rates, dead zones formed when heterogeneous nuclei on baffles grew into a sherbet-like unmixed slurry, and (2) at high stir rates, the incorporation of air into the slurry resulted in the accumulation of a stable foam that did not interact with the liquid slurry. These mixing issues limited determination of secondary nucleation kinetics to a modest range of conditions. Offline image analysis indicated that secondary nucleation is dominant over primary nucleation and that breakage of high aspect ratio crystals may play a role in crystal breeding. These kinetic data, PAT tools, and process insights are discussed in the context of a continuous manufacturing scenario.

Conceptual Validation of Stochastic and Deterministic Methods To Estimate Crystal Nucleation Rates

“…Recent experimental evidence indicates that secondary nucleation may be the dominant type of nucleation in unseeded batch processes for a wide variety of compounds, implying that this case is of practical relevance in industrial crystallization. 9,22,53,54 As demonstrated in section 4.1, deterministic methods in this case overpredict primary nucleation rates while stochastic ones underpredict them. Importantly, this conceptual finding is supported by a number of recent experimental studies in the literature, which observed that stochastic methods predicted smaller primary nucleation rates than deterministic methods for the same system.…”

Reactor Design and Optimization of α-Amino Ester Hydrolase- Catalyzed Synthesis of Cephalexin

“…A single CSTR is perhaps the simplest operation of an enzymatic reactor and has been simulated and operated for PGA previously ( McDonald et al, 2019a ; McDonald et al, 2019b ). Here, simulations involving AEH were performed by varying reactor residence times and inlet PGME and 7-ADCA concentrations from 25–1,000 mM and 25–500 mM respectively to study how conversion, fractional yield, and productivity are affected by easily tunable operating conditions.…”

“…Specifically, consumption of cephalosporins grew 94% between 2000 and 2010 and global production of cephalexin currently exceeds approximately 4,000 tons each year ( Laxminarayan 2014 ; Van Boeckel et al, 2014 ). Recent studies have focused specifically on developing a continuous operation in simulations of continuous reactor designs using both enzymatic reaction and crystallization kinetics ( Valencia et al, 2012 ; Encarnación-Gómez et al, 2016 ; McDonald et al, 2017 ; McDonald et al, 2019a ; McDonald et al, 2019b ; McDonald et al, 2019c ; Cuthbertson et al, 2019 ; Salami et al, 2020 ).…”

Direct Observation of Growth Rate Dispersion in the Enzymatic Reactive Crystallization of Ampicillin

“…There is evidence that the presence of other species, in this case the reactants 6-APA and PGME, can inhibit the growth of ampicillin crystals [29,30], which may lead to lack of growth below a critical supersaturation. The lack of growth is postulated to arise from the other species adsorbing to the edge of growth steps and preventing the step from advancing; halting the layer-by-layer growth mechanism [31][32][33]. The advance of edges along the length of the crystal is visible on some crystals in the videos provided with the Supplemental Material, although to be visible at this magnification they must be groups or bunches of growth steps.…”